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Whole exome sequencing identifies a novel FANCD2 gene splice site mutation associated with disease progression in chronic myeloid leukemia: Implication in targeted therapy of advanced phase CML. Absar Muhammad,Mahmood Amer,Akhtar Tanveer,Basit Sulman,Ramzan Khushnooda,Jameel Abid,Afzal Sibtain,Ullah Anhar,Qureshi Kulsoom,Alanazi Nawaf,Iqbal Zafar Pakistan journal of pharmaceutical sciences Tyrosine Kinase Inhibitors (TKIs) have significantly improved the clinical outcome of BCR-ABL+ Chronic Phase-Chronic Myeloid Leukemia (CP-CML). Nonetheless, approximately one-third of the CP-CML patient's progress to advanced phases of CML (accelerated and blast phase). Impaired DNA repair including mutations in Fanconi anemia (FA) pathway genes are responsible for progression of many cancers. Nevertheless, FA-pathways genes have never been reported in myeloid cancers. Hence, this study was aimed to discover DNA repair genes associated with CML progression. AP-CML patients were subjected to whole exome sequencing along with appropriate controls. A novel splice site FANCD2 mutation was detected. FANCD2 is a well-known FA-pathway gene with established role in DNA repair. This is first report of FA-pathway DNA repair genes in myeloid cancers that can serve as a novel marker of CML progression to clinically intervene CML progression. Further studies are needed to establish the functional role of FANCD2 in CML progression that can provide novel insights into CML pathogenesis. This study also indicates that a combination TKIs and Poly (ADP-ribose) polymerase (PARP) inhibitors like Olaparib (FDA approved anti-cancer drug for FA-pathway gene mutations) could improve the clinical outcome CML patients in accelerated and blast-crisis phases of the disease.
Genetic analysis of a Fanconi anemia case revealed the presence of FANCF mutation (exon 1;469>C-T) with implications to develop acute myeloid leukemia. Molecular biology reports BACKGROUND:Fanconi anemia (FA) is a rare genetic disorder and one of the most common inherited forms of aplastic anemia. FA is an autosomal recessive or X-linked genetic disorder that is characterized by typical physical malformations and haematopoietic anomalies. In most cases of FA, patients harbor homozygous or double heterozygous mutations in the FANCA (60-65%), FANCC (10-15%), FANCG (~ 10%), FANCD2 (3-6%) or FANCF (2%) genes in different ethnic populations, which leads to inherited bone marrow failure (IBMF). Hence, it is important to screen such mutations in correlation with clinical manifestations of FA in various ethnic populations. APPROACH:An 11 year old female pediatric patient of an East India family was presented with febrile illness, having thrombocytopenia with positive dengue IgM (Immunoglobulin M) and treated as a case of dengue hemorrhagic fever at the initial stage of diagnosis. Chromosomal breakage study was performed based on the abnormal physical examination, which showed 100% breaks, triradials, and quadrilaterals in mitomycin (MMC)-induced peripheral blood lymphocyte culture. Importantly, conventional cytogenetic assay in most of the bone marrow cells revealed an additional gain in chromosome 3q+ [46,XX,add(3)(q25)] and terminal loss in chr8p- [46,XX,del(8)(p23)], which might have a prognostic relevance in the outcomes of the FA patient. The bone marrow aspiration and biopsy were repeated and the results showed acute leukemia with 39% blast cells. Whole-genome sequencing analysis of the patient confirmed the presence of (exon 1; 496 > C-T) non-sense mutation leading to a truncated FANCF protein attributed to a stop codon at the amino acid position 166. CONCLUSION:The study reported the presence of a homozygous C-T exon 1 mutation in FANCF gene in the female pediatric patient from Odisha, India associated with FA. Furthermore, both parents were found to be carriers of FANCF gene mutation, as this allele was found to be in heterozygous state upon genome sequencing. The pathogenicity of the agent was robustly supported by the clinical phenotype and biochemical observations, wherein the patient eventually developed acute myeloid leukemia. The findings of the study infer the importance of early detection of FA and the associated mutations, which might lead to the development of acute myeloid leukemia. 10.1007/s11033-022-08071-z
The DNA-binding activity of USP1-associated factor 1 is required for efficient RAD51-mediated homologous DNA pairing and homology-directed DNA repair. Liang Fengshan,Miller Adam S,Tang Caroline,Maranon David,Williamson Elizabeth A,Hromas Robert,Wiese Claudia,Zhao Weixing,Sung Patrick,Kupfer Gary M The Journal of biological chemistry USP1-associated factor 1 (UAF1) is an integral component of the RAD51-associated protein 1 (RAD51AP1)-UAF1-ubiquitin-specific peptidase 1 (USP1) trimeric deubiquitinase complex. This complex acts on DNA-bound, monoubiquitinated Fanconi anemia complementation group D2 (FANCD2) protein in the Fanconi anemia pathway of the DNA damage response. Moreover, RAD51AP1 and UAF1 cooperate to enhance homologous DNA pairing mediated by the recombinase RAD51 in DNA repair via the homologous recombination (HR) pathway. However, whereas the DNA-binding activity of RAD51AP1 has been shown to be important for RAD51-mediated homologous DNA pairing and HR-mediated DNA repair, the role of DNA binding by UAF1 in these processes is unclear. We have isolated mutant UAF1 variants that are impaired in DNA binding and tested them together with RAD51AP1 in RAD51-mediated HR. This biochemical analysis revealed that the DNA-binding activity of UAF1 is indispensable for enhanced RAD51 recombinase activity within the context of the UAF1-RAD51AP1 complex. In cells, DNA-binding deficiency of UAF1 increased DNA damage sensitivity and impaired HR efficiency, suggesting that UAF1 and RAD51AP1 have coordinated roles in DNA binding during HR and DNA damage repair. Our findings show that even though UAF1's DNA-binding activity is redundant with that of RAD51AP1 in FANCD2 deubiquitination, it is required for efficient HR-mediated chromosome damage repair. 10.1074/jbc.RA120.013714
Mammalian INO80 chromatin remodeler cooperates with FANCM to mediate DNA interstrand crosslink-induced checkpoint activation and repair. Andreev Veselin,Hristova Rossitsa,Asparuhova Mila,Danovski Georgi,Stoynov Stoyno,Gospodinov Anastas DNA repair Chromatin regulators play crucial roles in the DNA damage response. While the chromatin changes needed for double-strand break repair and nucleotide excision repair have been intensely studied, the chromatin requirements of interstrand crosslink (ICL) repair have remained largely unexplored. Here, we studied the effect of silencing the INO80 chromatin remodeler subunits on the cellular response to ICLs. Cells depleted of Ino80 ATPase were more sensitive to mitomycin C (MMC) and defective in FANCD2 chromatin recruitment. Ino80-deficient cells displayed strongly reduced Chk1 phosphorylation after MMC treatment indicating impaired ATR-dependent DNA damage signaling, likely due to the significantly slower RPA foci formation which we observed in these cells. MMC treatment of cells silenced for FANCM - a protein required for ICL-induced checkpoint signaling, Ino80 or both genes simultaneously led to similar decreases in RPA phosphorylation suggesting that the two proteins were involved in the same checkpoint pathway. Co-immunoprecipitation data indicated that Ino80 and FANCM interact physically. Taken together our data demonstrate for the first time that the INO80 chromatin remodeler cooperates with FANCM to mediate ICL-induced checkpoint activation by promoting accumulation of RPA at the lesion sites. This constitutes a novel mechanism by which the INO80 chromatin remodeler participates in the repair of ICLs and genome integrity maintenance. 10.1016/j.dnarep.2018.12.007
Mitotic DNA Synthesis in Untransformed Human Cells Preserves Common Fragile Site Stability via a FANCD2-Driven Mechanism That Requires HELQ. Journal of molecular biology Faithful genome duplication is a challenging task for dividing mammalian cells, particularly under replication stress where timely resolution of late replication intermediates (LRIs) becomes crucial prior to cell division. In human cancer cells, mitotic DNA repair synthesis (MiDAS) is described as a final mechanism for the resolution of LRIs to avoid lethal chromosome mis-segregation. RAD52-driven MiDAS achieves this mission in part by generating gaps/breaks on metaphase chromosomes, which preferentially occur at common fragile sites (CFS). We previously demonstrated that a MiDAS mechanism also exists in untransformed and primary human cells, which is RAD52 independent but requires FANCD2. However, the properties of this form of MiDAS are not well understood. Here, we report that FANCD2-driven MiDAS in untransformed human cells: 1) requires a prerequisite step of FANCD2 mono-ubiquitination by a subset of Fanconi anemia (FA) proteins, 2) primarily acts to preserve CFS stability but not to prevent chromosome mis-segregation, and 3) depends on HELQ, which potentially functions at an early step. Hence, FANCD2-driven MiDAS in untransformed cells is built to protect CFS stability, whereas RAD52-driven MiDAS in cancer cells is likely adapted to prevent chromosome mis-segregation at the cost of CFS expression. Notably, we also identified a novel form of MiDAS, which surfaces to function when FANCD2 is absent in untransformed cells. Our findings substantiate the complex nature of MiDAS and a link between its deficiencies and the pathogenesis of FA, a human genetic disease. 10.1016/j.jmb.2023.168294
Structural basis of the fanconi anemia-associated mutations within the FANCA and FANCG complex. Jeong Eunyoung,Lee Seong-Gyu,Kim Hyun-Suk,Yang Jihyeon,Shin Jinwoo,Kim Youngran,Kim Jihan,Schärer Orlando D,Kim Youngjin,Yeo Jung-Eun,Kim Ho Min,Cho Yunje Nucleic acids research Monoubiquitination of the Fanconi anemia complementation group D2 (FANCD2) protein by the FA core ubiquitin ligase complex is the central event in the FA pathway. FANCA and FANCG play major roles in the nuclear localization of the FA core complex. Mutations of these two genes are the most frequently observed genetic alterations in FA patients, and most point mutations in FANCA are clustered in the C-terminal domain (CTD). To understand the basis of the FA-associated FANCA mutations, we determined the cryo-electron microscopy (EM) structures of Xenopus laevis FANCA alone at 3.35 Å and 3.46 Å resolution and two distinct FANCA-FANCG complexes at 4.59 and 4.84 Å resolution, respectively. The FANCA CTD adopts an arc-shaped solenoid structure that forms a pseudo-symmetric dimer through its outer surface. FA- and cancer-associated point mutations are widely distributed over the CTD. The two different complex structures capture independent interactions of FANCG with either FANCA C-terminal HEAT repeats, or the N-terminal region. We show that mutations that disturb either of these two interactions prevent the nuclear localization of FANCA, thereby leading to an FA pathway defect. The structure provides insights into the function of FANCA CTD, and provides a framework for understanding FA- and cancer-associated mutations. 10.1093/nar/gkaa062
FANCD2 protects genome stability by recruiting RNA processing enzymes to resolve R-loops during mild replication stress. Okamoto Yusuke,Abe Masako,Itaya Akiko,Tomida Junya,Ishiai Masamichi,Takaori-Kondo Akifumi,Taoka Masato,Isobe Toshiaki,Takata Minoru The FEBS journal R-loops, which consist of DNA : RNA hybrids and displaced single-strand DNA, are a major threat to genome stability. We have previously reported that a key Fanconi anemia protein, FANCD2, accumulates on large fragile genes during mild replication stress in a manner depending on R-loops. In this study, we found that FANCD2 suppresses R-loop levels. Furthermore, we identified FANCD2 interactions with RNA processing factors, including hnRNP U and DDX47. Our data suggest that FANCD2, which accumulates with R-loops in chromatin, recruits these factors and thereby promotes efficient processing of long RNA transcripts. This may lead to a reduction in transcription-replication collisions, as detected by PLA between PCNA and RNA Polymerase II, and hence, lowered R-loop levels. We propose that this mechanism might contribute to maintenance of genome stability during mild replication stress. 10.1111/febs.14700
Small-Molecule Inhibition of UBE2T/FANCL-Mediated Ubiquitylation in the Fanconi Anemia Pathway. ACS chemical biology The Fanconi anemia pathway orchestrates the repair of DNA interstrand cross-links and stalled replication forks. A key step in this pathway is UBE2T and FANCL-dependent monoubiquitylation of the FANCD2-FANCI complex. The Fanconi anemia pathway represents an attractive therapeutic target, because activation of this pathway has been linked to chemotherapy resistance in several cancers. However, to date, very few selective inhibitors of ubiquitin conjugation pathways are known. By using a high-throughput screen-compatible assay, we have identified a small-molecule inhibitor of UBE2T/FANCL-mediated FANCD2 monoubiquitylation that sensitizes cells to the DNA cross-linking agent, carboplatin. 10.1021/acschembio.9b00570
In vivo analysis of FANCD2 recruitment at meiotic DNA breaks in Caenorhabditis elegans. Germoglio Marcello,Valenti Anna,Gallo Ines,Forenza Chiara,Santonicola Pamela,Silva Nicola,Adamo Adele Scientific reports Fanconi Anemia is a rare genetic disease associated with DNA repair defects, congenital abnormalities and infertility. Most of FA pathway is evolutionary conserved, allowing dissection and mechanistic studies in simpler model systems such as Caenorhabditis elegans. In the present study, we employed C. elegans to better understand the role of FA group D2 (FANCD2) protein in vivo, a key player in promoting genome stability. We report that localization of FCD-2/FANCD2 is dynamic during meiotic prophase I and requires its heterodimeric partner FNCI-1/FANCI. Strikingly, we found that FCD-2 recruitment depends on SPO-11-induced double-strand breaks (DSBs) but not RAD-51-mediated strand invasion. Furthermore, exposure to DNA damage-inducing agents boosts FCD-2 recruitment on the chromatin. Finally, analysis of genetic interaction between FCD-2 and BRC-1 (the C. elegans orthologue of mammalian BRCA1) supports a role for these proteins in different DSB repair pathways. Collectively, we showed a direct involvement of FCD-2 at DSBs and speculate on its function in driving meiotic DNA repair. 10.1038/s41598-019-57096-1
Ubiquitination Causes Fanconi Anemia-Linked ID Complex Ring Formation. Cancer discovery Monoubiquitinated FANCI and FANCD2 constitute the ID complex, which forms a sliding clamp on DNA. 10.1158/2159-8290.CD-RW2020-043
Inhibition of Ubiquitin Specific Protease 1 Sensitizes Colorectal Cancer Cells to DNA-Damaging Chemotherapeutics. Xu Xin,Li Shaoyan,Cui Ximao,Han Kunkun,Wang Jun,Hou Xiaodan,Cui Long,He Songbing,Xiao Jiecheng,Yang Yili Frontiers in oncology Mutations and altered expression of deubiquitinating enzymes (DUBs) have been found associated with many human diseases including cancers. In this study, Ubiquitin specific protease 1 (USP1) expression was found significantly increased in some colorectal cancers (CRC). The elevated USP1 level was associated with short overall survival of patients and with advanced stages of cancers. In cultured CRC cells, knockdown of USP1 induced growth arrest at G/M of cell cycle and reduced the expression of anti-apoptotic proteins Bcl-2 and Mcl-1. Its knockdown also led to reduction of DNA-repair related substrates FANCD2 and ID1. Further investigations found that small molecular inhibitor of USP1 ML323 sensitized CRC cells to DNA-targeting chemotherapeutics, including doxorubicin, TOPI/II inhibitors, and PARP inhibitor, but not to 5-Fu. These results indicate that USP1 plays a critical in colorectal cancer cell survival and is a promising target for anti-colorectal cancer chemotherapy. Targeting USP1 may represent an effective strategy to regulate the DNA-repairing system. 10.3389/fonc.2019.01406
Fancd2os Reduces Testosterone Production by Inhibiting Steroidogenic Enzymes and Promoting Cellular Apoptosis in Murine Testicular Leydig Cells. Endocrinology and metabolism (Seoul, Korea) BACKGRUOUND:It is well-established that serum testosterone in men decreases with age, yet the underlying mechanism of this change remains elusive. METHODS:The expression patterns of Fancd2 opposite-strand (Fancd2os) in BALB/c male mice and testicular tissue derived cell lines (GC-1, GC-2, TM3, and TM4) were assessed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence. The Fancd2os-overexpressing or knockdown TM3 cells were constructed by infecting them with lentivirus particles and were used to evaluated the function of Fancd2os. The testosterone production was measured using enzyme linked immunosorbent assay (ELISA) and the steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) were analysed using RT-PCR. The apoptosis of TM3 cells induced by ultraviolet light or testicular tissues was detected using flow cytometry, Western blot or dUTP-biotin nick end labeling (TUNEL) assays. Pearson correlation analysis was used to assess the correlation between the Fancd2os expression and TUNEL-positive staining in mouse testicular Leydig cells. RESULTS:The Fancd2os protein was predominantly expressed in mouse testicular Leydig cells and its expression increased with age. Fancd2os overexpression inhibited testosterone levels in TM3 Leydig cells, whereas knockdown of Fancd2os elevated testosterone production. Fancd2os overexpression downregulated the levels of StAR, P450scc and 3β-HSD, while Fancd2os knockdown reversed this effect. Fancd2os overexpression promoted ultraviolet light-induced apoptosis of TM3 cells. In contrast, Fancd2os knockdown restrained apoptosis in TM3 cells. In vivo assays revealed that higher Fancd2os levels and mouse age were associated with increased apoptosis in Leydig cells and decreased serum testosterone levels. Pearson correlation analysis exhibited a strong positive correlation between the expression of Fancd2os and TUNEL-positive staining in mouse testicular Leydig cells. CONCLUSION:Our findings suggest that Fancd2os regulates testosterone synthesis via both steroidogenic enzymes and the apoptotic pathway. 10.3803/EnM.2022.1431
Acetylation modulates the Fanconi anemia pathway by protecting FAAP20 from ubiquitin-mediated proteasomal degradation. Nagareddy Bhavika,Khan Arafat,Kim Hyungjin The Journal of biological chemistry Fanconi anemia (FA) is a chromosome instability syndrome of children caused by inherited mutations in one of FA genes, which together constitute a DNA interstrand cross-link (ICL) repair, or the FA pathway. Monoubiquitination of Fanconi anemia group D2 protein (FANCD2) by the multisubunit ubiquitin E3 ligase, the FA core complex, is an obligate step in activation of the FA pathway, and its activity needs to be tightly regulated. FAAP20 is a key structural component of the FA core complex, and regulated proteolysis of FAAP20 mediated by prolyl isomerization and phosphorylation at a consensus phosphodegron motif is essential for preserving the integrity of the FA core complex, and thus FANCD2 monoubiquitination. However, how ubiquitin-dependent FAAP20 degradation is modulated to fine-tune FA pathway activation remains largely un-known. Here, we present evidence that FAAP20 is acetylated by the acetyltransferase p300/CBP on lysine 152, the key residue that when polyubiquitinated results in the degradation of FAAP20. Acetylation or mutation of the lysine residue stabilizes FAAP20 by preventing its ubiquitination, thereby protecting it from proteasome-dependent FAAP20 degradation. Consequently, disruption of the FAAP20 acetylation pathway impairs FANCD2 activation. Together, our study reveals a competition mechanism between ubiquitination and acetylation of a common lysine residue that controls FAAP20 stability and highlights a complex balancing between different posttranslational modifications as a way to refine the FA pathway signaling required for DNA ICL repair and genome stability. 10.1074/jbc.RA120.015288
DNA requirement in FANCD2 deubiquitination by USP1-UAF1-RAD51AP1 in the Fanconi anemia DNA damage response. Liang Fengshan,Miller Adam S,Longerich Simonne,Tang Caroline,Maranon David,Williamson Elizabeth A,Hromas Robert,Wiese Claudia,Kupfer Gary M,Sung Patrick Nature communications Fanconi anemia (FA) is a multigenic disease of bone marrow failure and cancer susceptibility stemming from a failure to remove DNA crosslinks and other chromosomal lesions. Within the FA DNA damage response pathway, DNA-dependent monoubiquitinaton of FANCD2 licenses downstream events, while timely FANCD2 deubiquitination serves to extinguish the response. Here, we show with reconstituted biochemical systems, which we developed, that efficient FANCD2 deubiquitination by the USP1-UAF1 complex is dependent on DNA and DNA binding by UAF1. Surprisingly, we find that the DNA binding activity of the UAF1-associated protein RAD51AP1 can substitute for that of UAF1 in FANCD2 deubiquitination in our biochemical system. We also reveal the importance of DNA binding by UAF1 and RAD51AP1 in FANCD2 deubiquitination in the cellular setting. Our results provide insights into a key step in the FA pathway and help define the multifaceted role of the USP1-UAF1-RAD51AP1 complex in DNA damage tolerance and genome repair. 10.1038/s41467-019-10408-5
UHRF1 is indispensable for meiotic sex chromosome inactivation and interacts with the DNA damage response pathway in mice†. Biology of reproduction During male meiosis, the constitutively unsynapsed XY chromosomes undergo meiotic sex chromosome inactivation (MSCI), and the DNA damage response (DDR) pathway is critical for MSCI establishment. Our previous study showed that UHRF1 (ubiquitin-like, with PHD and ring finger domains 1) deletion led to meiotic arrest and male infertility; however, the underlying mechanisms of UHRF1 in the regulation of meiosis remain unclear. Here, we report that UHRF1 is required for MSCI and cooperates with the DDR pathway in male meiosis. UHRF1-deficient spermatocytes display aberrant pairing and synapsis of homologous chromosomes during the pachytene stage. In addition, UHRF1 deficiency leads to aberrant recruitment of ATR and FANCD2 on the sex chromosomes and disrupts the diffusion of ATR to the XY chromatin. Furthermore, we show that UHRF1 acts as a cofactor of BRCA1 to facilitate the recruitment of DDR factors onto sex chromosomes for MSCI establishment. Accordingly, deletion of UHRF1 leads to the failure of meiotic silencing on sex chromosomes, resulting in meiotic arrest. In addition to our previous findings, the present study reveals that UHRF1 participates in MSCI, ensuring the progression of male meiosis. This suggests a multifunctional role of UHRF1 in the male germline. 10.1093/biolre/ioac054
Hedgehog/GLI1 Transcriptionally Regulates FANCD2 in Ovarian Tumor Cells: Its Inhibition Induces HR-Deficiency and Synergistic Lethality with PARP Inhibition. Neoplasia (New York, N.Y.) Ovarian cancer (OC) is one of the most lethal type of cancer in women due to a lack of effective targeted therapies and high rates of treatment resistance and disease recurrence. Recently Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown promise as chemotherapeutic agents; however, their efficacy is limited to a small fraction of patients with BRCA mutations. Here we show a novel function for the Hedgehog (Hh) transcription factor Glioma associated protein 1 (GLI1) in regulation of key Fanconi anemia (FA) gene, FANCD2 in OC cells. GLI1 inhibition in HR-proficient OC cells induces HR deficiency (BRCAness), replication stress and synergistic lethality when combined with PARP inhibition. Treatment of OC cells with combination of GLI1 and PARP inhibitors shows enhanced DNA damage, synergy in cytotoxicity, and strong in vivo anticancer responses. 10.1016/j.neo.2021.06.010
FANCD2-Associated Nuclease 1 Partially Compensates for the Lack of Exonuclease 1 in Mismatch Repair. Molecular and cellular biology Germline mutations in the mismatch repair (MMR) genes , , , and are linked to cancer of the colon and other organs, characterized by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in , encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes to MMR ; cells lacking both EXO1 and FAN1 have an MMR defect and display resistance to -methyl--nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, suggesting that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1. 10.1128/MCB.00303-21
A clickable melphalan for monitoring DNA interstrand crosslink accumulation and detecting ICL repair defects in Fanconi anemia patient cells. Nucleic acids research Fanconi anemia (FA) is a genetic disorder associated with developmental defects, bone marrow failure and cancer. The FA pathway is crucial for the repair of DNA interstrand crosslinks (ICLs). In this study, we have developed and characterized a new tool to investigate ICL repair: a clickable version of the crosslinking agent melphalan which we name click-melphalan. Our results demonstrate that click-melphalan is as effective as its unmodified counterpart in generating ICLs and associated toxicity. The lesions induced by click-melphalan can be detected in cells by post-labelling with a fluorescent reporter and quantified using flow cytometry. Since click-melphalan induces both ICLs and monoadducts, we generated click-mono-melphalan, which only induces monoadducts, in order to distinguish between the two types of DNA repair. By using both molecules, we show that FANCD2 knock-out cells are deficient in removing click-melphalan-induced lesions. We also found that these cells display a delay in repairing click-mono-melphalan-induced monoadducts. Our data further revealed that the presence of unrepaired ICLs inhibits monoadduct repair. Finally, our study demonstrates that these clickable molecules can differentiate intrinsic DNA repair deficiencies in primary FA patient cells from those in primary xeroderma pigmentosum patient cells. As such, these molecules may have potential for developing diagnostic tests. 10.1093/nar/gkad559
Structure of the FA core ubiquitin ligase closing the ID clamp on DNA. Nature structural & molecular biology The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand crosslinks. Central to the pathway is the FA core complex, a ubiquitin ligase of nine subunits that monoubiquitinates the FANCI-FANCD2 (ID) DNA clamp. The 3.1 Å structure of the 1.1-MDa human FA core complex, described here, reveals an asymmetric assembly with two copies of all but the FANCC, FANCE and FANCF subunits. The asymmetry is crucial, as it prevents the binding of a second FANCC-FANCE-FANCF subcomplex that inhibits the recruitment of the UBE2T ubiquitin conjugating enzyme, and instead creates an ID binding site. A single active site then ubiquitinates FANCD2 and FANCI sequentially. We also present the 4.2-Å structures of the human core-UBE2T-ID-DNA complex in three conformations captured during monoubiquitination. They reveal the core-UBE2T complex remodeling the ID-DNA complex, closing the clamp on the DNA before ubiquitination. Monoubiquitination then prevents clamp opening after release from the core. 10.1038/s41594-021-00568-8
SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability. EMBO reports SMC5/6 function in genome integrity remains elusive. Here, we show that SMC5 dysfunction in avian DT40 B cells causes mitotic delay and hypersensitivity toward DNA intra- and inter-strand crosslinkers (ICLs), with smc5 mutants being epistatic to FANCC and FANCM mutations affecting the Fanconi anemia (FA) pathway. Mutations in the checkpoint clamp loader RAD17 and the DNA helicase DDX11, acting in an FA-like pathway, do not aggravate the damage sensitivity caused by SMC5 dysfunction in DT40 cells. SMC5/6 knockdown in HeLa cells causes MMC sensitivity, increases nuclear bridges, micronuclei, and mitotic catastrophes in a manner similar and non-additive to FANCD2 knockdown. In both DT40 and HeLa systems, SMC5/6 deficiency does not affect FANCD2 ubiquitylation and, unlike FANCD2 depletion, RAD51 focus formation. SMC5/6 components further physically interact with FANCD2-I in human cells. Altogether, our data suggest that SMC5/6 functions jointly with the FA pathway to support genome integrity and DNA repair and may be implicated in FA or FA-related human disorders. 10.15252/embr.201948222
[Diagnosis status and genetic characteristics analysis of Fanconi anemia in China]. Zhonghua er ke za zhi = Chinese journal of pediatrics To analyze the clinical and molecular diagnostic status of Fanconi anemia (FA) in China. The General situation, clinical manifestations and chromosome breakage test and genetic test results of 107 pediatric FA cases registered in the Chinese Blood and Marrow Transplantation Registry Group (CBMTRG) and the Chinese Children Blood and Marrow Transplantation Registry Group (CCBMTRG) from August 2009 to January 2022 were analyzed retrospectively. Children with FANCA gene variants were divided into mild and severe groups based on the type of variant, and Wilcoxon-test was used to compare the phenotypic differences between groups. Of the 176 registered FA patients, 69 (39.2%) cases were excluded due to lack of definitive genetic diagnosis results, and the remaining 107 children from 15 hospitals were included in the study, including 70 males and 37 females. The age at transplantation treatment were 6 (4, 9) years. The enrolled children were involved in 10 pathogenic genes, including 89 cases of FANCA gene, 7 cases of FANCG gene, 3 cases of FANCB gene, 2 cases of FANCE gene and 1 case each of FANCC, FANCD1, FANCD2, FANCF, FANCJ, and FANCN gene. Compound heterozygous or homozygous of loss-of-function variants account for 69.2% (72/104). Loss-of-function variants account for 79.2% (141/178) in FANCA gene variants, and 20.8% (37/178) were large exon deletions. Fifty-five children (51.4%) had chromosome breakage test records, with a positive rate of 81.8% (45/55). There were 172 congenital malformations in 80 children.Café-au-Lait spots (16.3%, 28/172), thumb deformities (16.3%,28/172), polydactyly (13.9%, 24/172), and short stature (12.2%, 21/172) were the most common congenital malformations in Chinese children with FA. No significant difference was found in the number of congenital malformations between children with severe (50 cases) and mild FANCA variants (26 cases) (=-1.33, =0.185). FANCA gene is the main pathogenic gene in children with FA, where the detection of its exon deletion should be strengthened clinically. There were no phenotypic differences among children with different types of FANCA variants. Chromosome break test is helpful to determine the pathogenicity of variants, but its accuracy needs to be improved. 10.3760/cma.j.cn112140-20230606-00383
Generating New FANCA-Deficient HNSCC Cell Lines by Genomic Editing Recapitulates the Cellular Phenotypes of Fanconi Anemia. Genes Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, -mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These -mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with mutations are phenocopies of FA-HNSCC cells. 10.3390/genes12040548
Fanconi anemia protein FANCI functions in ribosome biogenesis. Proceedings of the National Academy of Sciences of the United States of America Fanconi anemia (FA) is a disease of DNA repair characterized by bone marrow failure and a reduced ability to remove DNA interstrand cross-links. Here, we provide evidence that the FA protein FANCI also functions in ribosome biogenesis, the process of making ribosomes that initiates in the nucleolus. We show that FANCI localizes to the nucleolus and is functionally and physically tied to the transcription of pre-ribosomal RNA (pre-rRNA) and to large ribosomal subunit (LSU) pre-rRNA processing independent of FANCD2. While FANCI is known to be monoubiquitinated when activated for DNA repair, we find that it is predominantly in the deubiquitinated state in the nucleolus, requiring the nucleoplasmic deubiquitinase (DUB) USP1 and the nucleolar DUB USP36. Our model suggests a possible dual pathophysiology for FA that includes defects in DNA repair and in ribosome biogenesis. 10.1073/pnas.1811557116
Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery. Raghunandan Maya,Yeo Jung Eun,Walter Ryan,Saito Kai,Harvey Adam J,Ittershagen Stacie,Lee Eun-A,Yang Jihyeon,Hoatlin Maureen E,Bielinsky Anja K,Hendrickson Eric A,Schärer Orlando,Sobeck Alexandra Human molecular genetics Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Specifically, the FA pathway functions to protect genome stability during DNA replication. The central FA pathway protein, FANCD2, locates to stalled replication forks and recruits homologous recombination (HR) factors such as CtBP interacting protein (CtIP) to promote replication fork restart while suppressing new origin firing. Here, we identify alpha-thalassemia retardation syndrome X-linked (ATRX) as a novel physical and functional interaction partner of FANCD2. ATRX is a chromatin remodeler that forms a complex with Death domain-associated protein 6 (DAXX) to deposit the histone variant H3.3 into specific genomic regions. Intriguingly, ATRX was recently implicated in replication fork recovery; however, the underlying mechanism(s) remained incompletely understood. Our findings demonstrate that ATRX forms a constitutive protein complex with FANCD2 and protects FANCD2 from proteasomal degradation. ATRX and FANCD2 localize to stalled replication forks where they cooperate to recruit CtIP and promote MRE11 exonuclease-dependent fork restart while suppressing the firing of new replication origins. Remarkably, replication restart requires the concerted histone H3 chaperone activities of ATRX/DAXX and FANCD2, demonstrating that coordinated histone H3 variant deposition is a crucial event during the reinitiation of replicative DNA synthesis. Lastly, ATRX also cooperates with FANCD2 to promote the HR-dependent repair of directly induced DNA double-stranded breaks. We propose that ATRX is a novel functional partner of FANCD2 to promote histone deposition-dependent HR mechanisms in S-phase. 10.1093/hmg/ddz250
FANCI functions as a repair/apoptosis switch in response to DNA crosslinks. Developmental cell Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair. 10.1016/j.devcel.2021.06.010
Structure of the Fanconi anaemia monoubiquitin ligase complex. Nature The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair. 10.1038/s41586-019-1703-4
HPV Enhances HNSCC Chemosensitization by Inhibiting SERPINB3 Expression to Disrupt the Fanconi Anemia Pathway. Advanced science (Weinheim, Baden-Wurttemberg, Germany) Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck, and the prognosis of patients is poor due to chemotherapeutic resistance. Interestingly, patients with HNSCC induced by human papillomavirus (HPV) infection are more sensitive to chemotherapy and display a better prognosis than HPV-negative patients. The biological relevance of HPV infection and the mechanism underlying chemosensitivity to cisplatin remain unknown. Herein, SERPINB3 is identified as an important target for regulation of cisplatin sensitivity by HPV-E6/E7 in HNSCC. Downregulation of SERPINB3 inhibits cisplatin-induced DNA damage repair and enhances the cytotoxicity of cisplatin. In detail, decreasing SERPINB3 expression reduces the USP1-mediated deubiquitination of FANCD2-FANCI in the Fanconi anemia pathway, thereby interfering with cisplatin-induced DNA interstrand crosslinks repair and further contributing to HNSCC cell apoptosis. To translate this finding, pH-responsive nanoparticles are used to deliver SERPINB3 small interfering RNA in combination with cisplatin, and this treatment successfully reverses cisplatin chemotherapeutic resistance in a patient-derived xenograft model from HPV-negative HNSCC. Taken together, these findings suggest that targeting SERPINB3 based on HPV-positive HNSCC is a potential strategy to overcome cisplatin resistance in HPV-negative HNSCC and improves the prognosis of this disease. 10.1002/advs.202202437
Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2. Cell reports Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following introduction of genomic ICLs and dependent on ATR and FANCD2 but not on the Fanconi anemia core proteins or FAAP24, FANCM binds the replisome complex, with concomitant release of the GINS proteins. In situ analysis of replisomes proximal to ICLs confirms the ATR-dependent release of GINS proteins while CDC45 is retained on the remodeled replisome. The results demonstrate the plasticity of CMG composition in response to replication stress. 10.1016/j.celrep.2019.04.032
FANCI plays an essential role in spermatogenesis and regulates meiotic histone methylation. Cell death & disease FANCI is an essential component of Fanconi anemia pathway, which is responsible for the repair of DNA interstrand cross-links (ICLs). As an evolutionarily related partner of FANCD2, FANCI functions together with FANCD2 downstream of FA core complex. Currently, growing evidences showed that the essential role of FA pathway in male fertility. However, the underlying mechanisms for FANCI in regulating spermatogenesis remain unclear. In the present study, we found that the male Fanci mice were sterile and exhibited abnormal spermatogenesis, including massive germ cell apoptosis in seminiferous tubules and dramatically decreased number of sperms in epididymis. Besides, FANCI deletion impaired maintenance of undifferentiated spermatogonia. Further investigation indicated that FANCI was essential for FANCD2 foci formation and regulated H3K4 and H3K9 methylation on meiotic sex chromosomes. These findings elucidate the role and mechanism of FANCI during spermatogenesis in mice and provide new insights into the etiology and molecular basis of nonobstructive azoospermia. 10.1038/s41419-021-04034-7
Association of clinical severity with FANCB variant type in Fanconi anemia. Blood Fanconi anemia (FA) is the most common genetic cause of bone marrow failure and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry. Those with FANCB deletion or truncation demonstrate earlier-than-average onset of bone marrow failure and more severe congenital abnormalities compared with a large series of FA individuals in published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization were associated with 2 missense variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays tended to show earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is associated with level of residual FANCD2 monoubiquitination. 10.1182/blood.2019003249
High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2. Khanal Sujita,Galloway Denise A PLoS pathogens Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-strand breaks (DSB), which subsequently hindered the recruitment of the downstream protein Rad51 to DSB in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for effective ICL repair. Delayed FancD2 de-ubiquitination was associated with the increased chromatin retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the response to cisplatin therapies. 10.1371/journal.ppat.1007442
Inactivation of ribosomal protein S27-like impairs DNA interstrand cross-link repair by destabilization of FANCD2 and FANCI. Sun Siyuan,He Hengqian,Ma Yuanyuan,Xu Jie,Chen Guoan,Sun Yi,Xiong Xiufang Cell death & disease Ribosomal protein S27-like (RPS27L), an evolutionarily conserved ribosomal protein and a direct p53 target, plays an important role in maintenance of genome integrity. We have previously reported that RPS27L regulates radiation sensitivity via the MDM2-p53 and MDM2-MRN-ATM axes. Whether and how RPS27L modulates DNA interstrand cross-link (ICL) repair is unknown. Here we identified that RPS27L binds to FANCD2 and FANCI, two Fanconi anemia (FA) proteins functioning in ICL repair pathway. Upon RPS27L knockdown, the levels of FANCD2 and FANCI are reduced due to accelerated degradation via p62-mediated autophagy-lysosome pathway, which is abrogated by chloroquine (CQ) treatment or Beclin 1 knockdown. Biologically, RPS27L knockdown suppresses FANCD2 foci formation and impairs ICL repair upon exposure to ICL-inducing agent mitomycin C (MMC) in lung cancer cells. This effect of MMC sensitization can be partially reversed by CQ treatment. Together, our study shows that RPS27L positively regulates ICL repair by binding with FANCD2 and FANCI to prevent their degradation via autophagy-lysosome system. 10.1038/s41419-020-03082-9
FAN1's protection against CGG repeat expansion requires its nuclease activity and is FANCD2-independent. Nucleic acids research The Repeat Expansion Diseases, a large group of human diseases that includes the fragile X-related disorders (FXDs) and Huntington's disease (HD), all result from expansion of a disease-specific microsatellite via a mechanism that is not fully understood. We have previously shown that mismatch repair (MMR) proteins are required for expansion in a mouse model of the FXDs, but that the FANCD2 and FANCI associated nuclease 1 (FAN1), a component of the Fanconi anemia (FA) DNA repair pathway, is protective. FAN1's nuclease activity has been reported to be dispensable for protection against expansion in an HD cell model. However, we show here that in a FXD mouse model a point mutation in the nuclease domain of FAN1 has the same effect on expansion as a null mutation. Furthermore, we show that FAN1 and another nuclease, EXO1, have an additive effect in protecting against MSH3-dependent expansions. Lastly, we show that the loss of FANCD2, a vital component of the Fanconi anemia DNA repair pathway, has no effect on expansions. Thus, FAN1 protects against MSH3-dependent expansions without diverting the expansion intermediates into the canonical FA pathway and this protection depends on FAN1 having an intact nuclease domain. 10.1093/nar/gkab899
FANCD2 limits acetaldehyde-induced genomic instability during DNA replication in esophageal keratinocytes. Molecular oncology Individuals with Fanconi anemia (FA), a rare genetic bone marrow failure syndrome, have an increased risk of young-onset head and neck squamous cell carcinomas (SCCs) and esophageal SCC. The FA DNA repair pathway is activated upon DNA damage induced by acetaldehyde, a chief alcohol metabolite and one of the major carcinogens in humans. However, the molecular basis of acetaldehyde-induced genomic instability in SCCs of the head and neck and of the esophagus in FA remains elusive. Here, we report the effects of acetaldehyde on replication stress response in esophageal epithelial cells (keratinocytes). Acetaldehyde-exposed esophageal keratinocytes displayed accumulation of DNA damage foci consisting of 53BP1 and BRCA1. At physiologically relevant concentrations, acetaldehyde activated the ATR-Chk1 pathway, leading to S- and G2/M-phase delay with accumulation of the FA complementation group D2 protein (FANCD2) at the sites of DNA synthesis, suggesting that acetaldehyde impedes replication fork progression. Consistently, depletion of the replication fork protection protein Timeless led to elevated DNA damage upon acetaldehyde exposure. Furthermore, FANCD2 depletion exacerbated replication abnormalities, elevated DNA damage, and led to apoptotic cell death, indicating that FANCD2 prevents acetaldehyde-induced genomic instability in esophageal keratinocytes. These observations contribute to our understanding of the mechanisms that drive genomic instability in FA patients and alcohol-related carcinogenesis, thereby providing a translational implication in the development of more effective therapies for SCCs. 10.1002/1878-0261.13072
Silencing of FANCI Promotes DNA Damage and Sensitizes Ovarian Cancer Cells to Carboplatin. Current cancer drug targets BACKGROUND:Ovarian cancer (OVCA) has unique epigenetic alterations and defects in homologous recombination (HR). Despite initial sensitivity to platinum-based chemotherapy, HR dysfunctional tumors eventually acquire drug resistance. Fanconi anemia (FA) is characterized by bone marrow failure (BMF) and a reduced ability to eradicate DNA interstrand cross-links (ICL). However, the mechanism of chemoresistance mediated by FANCI was unclear in OVCA. OBJECTIVE:We explore to identify whether FANCI was involved in chemoresistance in OVCA. METHODS:FANCI expression and epigenetic alterations were analyzed, respectively, using TIMER and cBioPortal. The correlation between FANCI expression and the survival of OVCA patients was analyzed using Kaplan-Meier Plotter, GSE63885, and TCGA-OVCA dataset. FANCI expression in OVCA was detected by immunohistochemistry. Cell proliferation, migration, and invasion in FANCI inhibiting cells were assessed by CCK-8 and Transwell. Apoptosis and DNA damage were examined by flow cytometry and immunofluorescence. Meanwhile, the activity of caspase 3/7 was detected by Caspase-Glo® 3/7 kit. In addition, the expression of FANCI, γH2AX, and apoptosis effectors was examined by Western blot. RESULTS:FANCI has copy number variations (CNVs) in OVCA. The high expression of FANCI in OVCA patients was associated with poor survival. Moreover, FANCI expression was correlated with the response to chemotherapy in OVCA. FANCI expression in OVCA cells was induced by carboplatin in a time-dependent manner. Silencing of FANCI had no effect on cell proliferation, but hindered OVCA cell migration and invasion. Mechanically, knockdown of FANCI enhanced DNA damage-induced apoptosis through the CHK1/2-P53-P21 pathway. CONCLUSION:FANCI may be a potential therapeutic target for OVCA patients. 10.2174/1568009622666220331091709
Monoubiquitination by the human Fanconi anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays. eLife FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show using electron microscopy that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding. 10.7554/eLife.54128
FANCD2 knockdown with shRNA interference enhances the ionizing radiation sensitivity of nasopharyngeal carcinoma CNE-2 cells. Bao Yilin,Feng Huajun,Zhao Feipeng,Zhang Lijun,Xu Shengen,Zhang Conghong,Zhao Chong,Qin Gang Neoplasma Fanconi anemia complementation group D2 (FANCD2) has been associated with the sensitivity of tumor cells to DNA crosslinking damaging agents in certain solid tumors. However, its role in nasopharyngeal carcinoma (NPC) is still unclear. In the present study, the role of FANCD2 in the response of NPC CNE-2 cells to radiation was investigated. A CNE-2 cell model with stable FANCD2 silencing was constructed by lentiviral transfection. Fluorescence quantitative PCR and western blotting were used to evaluate FANCD2 expression in CNE-2 cells. The biological impact of FANCD2 silencing on the response of CNE-2 cells to radiation was investigated in vitro and in vivo. The microarray technology, western blotting, and immunohistochemistry were used to analyze the proteins involved in related pathways after irradiation to investigate the underlying mechanism. Lentivirus-mediated shRNA interference stably silenced the FANCD2 gene in CNE-2 cells. In vitro, in the FANCD2-silenced group, cell proliferation was significantly inhibited, apoptosis was increased, and the cell cycle was arrested at the G2/M phase after irradiation. In vivo, FANCD2 silencing slowed tumor growth, as the volume and weight of the xenograft tumors were significantly decreased. Both in vitro and in vivo, the differentially expressed genes NUPR1, FLI1, and FGF21 were downregulated in the FANCD2-silenced group. Our results show that FANCD2 silencing affected the sensitivity of CNE-2 cells to ionizing radiation by regulating cell proliferation, apoptosis, and cell cycle distribution. The mechanism might be associated with changes in NUPR1, FLI1, and FGF21 protein expression due to the FANCD2 silencing. This study provides a promising target for NPC radiotherapy. 10.4149/neo_2020_200511N516
ERCC1-XPF targeting to psoralen-DNA crosslinks depends on XPA and FANCD2. Cellular and molecular life sciences : CMLS The effectiveness of many DNA-damaging chemotherapeutic drugs depends on their ability to form monoadducts, intrastrand crosslinks and/or interstrand crosslinks (ICLs) that interfere with transcription and replication. The ERCC1-XPF endonuclease plays a critical role in removal of these lesions by incising DNA either as part of nucleotide excision repair (NER) or interstrand crosslink repair (ICLR). Engagement of ERCC1-XPF in NER is well characterized and is facilitated by binding to the XPA protein. However, ERCC1-XPF recruitment to ICLs is less well understood. Moreover, specific mutations in XPF have been found to disrupt its function in ICLR but not in NER, but whether this involves differences in lesion targeting is unknown. Here, we imaged GFP-tagged ERCC1, XPF and ICLR-defective XPF mutants to investigate how in human cells ERCC1-XPF is localized to different types of psoralen-induced DNA lesions, repaired by either NER or ICLR. Our results confirm its dependence on XPA in NER and furthermore show that its engagement in ICLR is dependent on FANCD2. Interestingly, we find that two ICLR-defective XPF mutants (R689S and S786F) are less well recruited to ICLs. These studies highlight the differential mechanisms that regulate ERCC1-XPF activity in DNA repair. 10.1007/s00018-019-03264-5
Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA. EMBO reports The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double-stranded DNA via promoting a large-scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary "Arm" ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA. 10.15252/embr.202050133
FANCD2 Confers a Malignant Phenotype in Esophageal Squamous Cell Carcinoma by Regulating Cell Cycle Progression. Lei Lisa Chan,Yu Valen Zhuoyou,Ko Josephine Mun Yee,Ning Lvwen,Lung Maria Li Cancers Fanconi anemia patients with germline genetic defects in are highly susceptible to cancers. Esophageal squamous cell carcinoma (ESCC) is a deadly cancer. Little is known about the function of in ESCC. For detailed molecular and mechanistic insights on the functional role of in ESCC, in vivo and in vitro assays and RNA sequencing approaches were used. Utilizing Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology, knockout models were established to examine the functional impact in mouse models for tumor growth and metastasis and in vitro assays for cell growth, cell cycle, and cellular localization. Our RNA sequence analyses were integrated with public datasets. confers a malignant phenotype in ESCC. is significantly upregulated in ESCC tumors, as compared to normal counterparts. Depletion of FANCD2 protein expression significantly suppresses the cancer cell proliferation and tumor colony formation and metastasis potential, as well as cell cycle progression, by involving cyclin-CDK and ATR/ATM signaling. FANCD2 translocates from the nucleus to the cytoplasm during cell cycle progression. We provide evidence of a novel role of in ESCC tumor progression and its potential usefulness as a biomarker for ESCC disease management. 10.3390/cancers12092545
Fanconi anemia DNA crosslink repair factors protect against LINE-1 retrotransposition during mouse development. Nature structural & molecular biology Long interspersed nuclear element 1 (LINE-1) is the only autonomous retrotransposon in humans and new integrations are a major source of genetic variation between individuals. These events can also lead to de novo germline mutations, giving rise to heritable genetic diseases. Recently, a role for DNA repair in regulating these events has been identified. Here we find that Fanconi anemia (FA) DNA crosslink repair factors act in a common pathway to prevent retrotransposition. We purify recombinant SLX4-XPF-ERCC1, the crosslink repair incision complex, and find that it cleaves putative nucleic acid intermediates of retrotransposition. Mice deficient in upstream crosslink repair signaling (FANCA), a downstream component (FANCD2) or the nuclease XPF-ERCC1 show increased LINE-1 retrotransposition in vivo. Organisms limit retrotransposition through transcriptional silencing but this protection is attenuated during early development leaving the zygote vulnerable. We find that during this window of vulnerability, DNA crosslink repair acts as a failsafe to prevent retrotransposition. Together, our results indicate that the FA DNA crosslink repair pathway acts together to protect against mutation by restricting LINE-1 retrotransposition. 10.1038/s41594-023-01067-8
ATM participates in fine particulate matter-induced airway inflammation through regulating DNA damage and DNA damage response. Environmental toxicology The relationship between fine particulate matter (PM2.5) and chronic airway inflammatory diseases, such as chronic obstructive pulmonary disease and asthma, have garnered public attention, while the detailed mechanisms of PM2.5-induced airway inflammation remain unclear. This study reveals that PM2.5 induces airway inflammation both in vivo and in vitro, and, moreover, identifies DNA damage and DNA damage repair (DDR) as results of this exposure. Ataxia telangiectasia-mutated heterozygous (ATM ) and wild-type C57BL/6 (WT) mice were exposed to PM2.5. The results show that, following exposure to PM2.5, the number of neutrophils in broncho alveolar lavage fluid and the mRNA expression of CXCL-1 in lung tissues of the ATM mice were lower than those of the WT mice. The mRNA expression of FANCD2 and FANCI were also down-regulated. Human bronchial epithelial (HBE) cells were transfected with ATM-siRNA to induce down-regulation of ATM gene expression and were subsequently stimulated with PM2.5. The results show that the mRNA expression of TNF-α decreased in the ATM-siRNA-transfected cells. The mRNA expression of CXCL-1 and CXCL-2 in peritoneal macrophages, derived from ATM-null mice in which experiments showed that the protein expression of FANCD2 and FANCI decreased, were also decreased after PM2.5 exposure in ATM-siRNA-transfected HBE cells. In conclusion, PM2.5-induced airway inflammation is alleviated in ATM mice compared with WT mice. ATM promotes PM2.5-induced airway inflammation, which may be attributed to the regulation of DNA damage and DDR. 10.1002/tox.23901
DNA clamp function of the monoubiquitinated Fanconi anaemia ID complex. Wang Renjing,Wang Shengliu,Dhar Ankita,Peralta Christopher,Pavletich Nikola P Nature The ID complex, involving the proteins FANCI and FANCD2, is required for the repair of DNA interstrand crosslinks (ICL) and related lesions. These proteins are mutated in Fanconi anaemia, a disease in which patients are predisposed to cancer. The Fanconi anaemia pathway of ICL repair is activated when a replication fork stalls at an ICL; this triggers monoubiquitination of the ID complex, in which one ubiquitin molecule is conjugated to each of FANCI and FANCD2. Monoubiquitination of ID is essential for ICL repair by excision, translesion synthesis and homologous recombination; however, its function remains unknown. Here we report a cryo-electron microscopy structure of the monoubiquitinated human ID complex bound to DNA, and reveal that it forms a closed ring that encircles the DNA. By comparison with the structure of the non-ubiquitinated ID complex bound to ICL DNA-which we also report here-we show that monoubiquitination triggers a complete rearrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. These structures-together with biochemical data-indicate that the monoubiquitinated ID complex loses its preference for ICL and related branched DNA structures, and becomes a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how monoubiquitination in general can induce an alternative protein structure with a new function. 10.1038/s41586-020-2110-6
A Fanca knockout mouse model reveals novel Fancd2 function. Biochemical and biophysical research communications Fanconi anemia (FA) is a genetically and clinically heterogenous inherited disorder. Clinically, Fanca subtype patients exhibited milder phenotypes compared to Fancd2 subtypes. Increasing evidence suggests that Fancd2 perform independent functions, but the detailed mechanisms are not well characterized. In this study, we developed a Fanca KO mice model in C57BL/6 background with ATG region deletion, then performed a detailed FA phenotypes characterization and analysis with Fanca KO mice and Fancd2 KO mice in the same congenic background. We found that both the Fanca KO and Fancd2 KO cause severe FA phenotypes in mice. However, Fanca KO mice exhibited milder FA phenotypes comparing to Fancd2 KO mice. Fanca KO mice showed higher embryonic and postnatal survival rate, less congenital eye defects in early development. At adult stage, Fanca KO mice showed increased HSC number and reconstitution function. Furthermore, we did RNA-seq study and identified differential expression of Dlk1 and Dlk1 pathway genes in Fanca KO and Fancd2 KO embryonic cells and adult HSCs. Finally, we revealed that Fancd2 was expressed and physically interact with Dlk1 in Fanca KO cells. Collectively, our findings suggested that Fancd2 has distinct functions in the absence of Fanca. 10.1016/j.bbrc.2023.149454
Fanconi anemia and mTOR pathways functionally interact during stalled replication fork recovery. FEBS letters We have previously demonstrated that Fanconi anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous studies suggest a connection between the FA protein FANCD2 and the non-FA protein mechanistic target of rapamycin (mTOR). A recent study showed that mTOR is involved in actin-dependent DNA replication fork restart, suggesting possible roles in the FA DNA repair pathway. In this study, we demonstrate that during replication stress mTOR interacts and cooperates with FANCD2 to provide cellular stability, mediate stalled replication fork restart, and prevent nucleolytic degradation of the nascent DNA strands. Taken together, this study unravels a novel functional cross-talk between two important mechanisms: mTOR and FA DNA repair pathways that ensure genomic stability. 10.1002/1873-3468.14035
A comprehensive analysis of Fanconi anemia genes in Chinese patients with high-risk hereditary breast cancer. Journal of cancer research and clinical oncology BACKGROUND:Four Fanconi anemia (FA) genes (BRCA1, BRCA2, PALB2 and RAD51C) are defined as breast cancer (BC) susceptibility genes. Other FA genes have been inconsistently associated with BC. Thus, the role of other FA genes in BC should be explored in specific populations. METHODS:Mutations in 16 FA genes were screened with a 98-gene panel sequencing assay in a cohort of 1481 Chinese patients with high-risk hereditary BC. The association between mutations and clinicopathological characteristics as well as prognosis was analyzed. The risk of BC in carriers of FA gene mutations was assessed in the Genome Aggregation Database and the Westlake Biobank for Chinese cohort. RESULTS:A total of 2.57% (38/1481) BC patients were identified who had 12 other FA gene germline mutations. Among them, the most frequently mutated gene was FANCA (8/1481, 0.54%). These 38 patients carried 35 distinct pathogenic/likely pathogenic variants, of which 21 were novel. We found one rare FANCB deleterious variant (c.1327-3dupT) in our cohort. There was a statistically significant difference in lymph node status between FA gene mutation carriers and non-carriers (p = 0.041). We observed a trend that mutation carriers had larger tumor sizes, lower estrogen receptor (ER) and progesterone receptor (PR) positivity rates, and lower 3.5-year invasive disease-free survival (iDFS) and distant recurrence-free survival (DRFS) rates than non-carriers (tumor size > 2 cm: 51.43% vs. 45.63%; ER positivity rates: 51.43% vs. 60.81%; PR positivity rates: 48.57% vs. 55.16%; 3.5-year iDFS rates: 58.8% vs. 66.7%; 3.5-year DRFS rates: 58.8% vs. 68.8%). The frequency of the mutations in FANCD2, FANCM and BRIP1 trended to be higher among BC cases than that in controls (p = 0.055, 0.08 and 0.08, respectively). CONCLUSION:This study comprehensively estimated the prevalence, clinicopathological characteristics, prognosis and risk of BC associated with deleterious variants in FA genes in Chinese high-risk hereditary BC patients. It enriches our understanding of the role of FA genes with BC. 10.1007/s00432-023-05236-6
Pan-cancer analysis of the tumorigenic role of Fanconi anemia complementation group D2 (FANCD2) in human tumors. Genomics Monoubiquitination of FANCD2 is a central step in the activation of the Fanconi anemia (FA) pathway after DNA damage. Defects in the FA pathway centered around FANCD2 not only lead to genomic instability but also induce tumorigenesis. At present, few studies have investigated FANCD2 in tumors, and no pan-cancer research on FANCD2 has been conducted. We conducted a comprehensive analysis of the role of FANCD2 in cancer using public databases and other published studies. Moreover, we evaluated the role of FANCD2 in the proliferation, migration and invasion of lung adenocarcinoma cells through in vitro and in vivo experiments, and explored the role of FANCD2 in cisplatin chemoresistance. We investigated the regulatory effect of FANCD2 on the cell cycle of lung adenocarcinoma cells by flow cytometry, and verified this effect by western blotting. FANCD2 expression is elevated in most TCGA tumors and shows a strong positive correlation with poor prognosis in tumor patients. In addition, FANCD2 expression shows strong correlations with immune infiltration, immune checkpoints, the tumor mutation burden (TMB), and microsatellite instability (MSI), which are immune-related features, suggesting that it may be a potential target of tumor immunotherapy. We further found that FANCD2 significantly promotes the proliferation, invasion, and migration abilities of lung adenocarcinoma cells and that its ability to promote cancer cell proliferation may be achieved by modulating the cell cycle. The findings indicate that FANCD2 is a potential biomarker and therapeutic target in cancer treatment by analyzing the oncogenic role of FANCD2 in different tumors. 10.1016/j.ygeno.2023.110762
UBE2T resolves transcription-replication conflicts and protects common fragile sites in primordial germ cells. Cellular and molecular life sciences : CMLS The proper development of primordial germ cells (PGCs) is an essential prerequisite for gametogenesis and mammalian fertility. The Fanconi anemia (FA) pathway functions in maintaining the development of PGCs. FANCT/UBE2T serves as an E2 ubiquitin-conjugating enzyme that ubiquitylates the FANCD2-FANCI complex to activate the FA pathway, but its role in the development of PGCs is not clear. In this study, we found that Ube2t knockout mice showed defects in PGC proliferation, leading to severe loss of germ cells after birth. Deletion of UBE2T exacerbated DNA damage and triggered the activation of the p53 pathway. We further demonstrated that UBE2T counteracted transcription-replication conflicts by resolving R-loops and stabilizing replication forks, and also protected common fragile sites by resolving R-loops in large genes and promoting mitotic DNA synthesis to maintain the genome stability of PGCs. Overall, these results provide new insights into the function and regulatory mechanisms of the FA pathway ensuring normal development of PGCs. 10.1007/s00018-023-04733-8
Class I HDAC Inhibition Leads to a Downregulation of FANCD2 and RAD51, and the Eradication of Glioblastoma Cells. Journal of personalized medicine HDAC inhibitors (HDACi) hold great potential as anticancer therapies due to their ability to regulate the acetylation of both histone and non-histone proteins, which is frequently disrupted in cancer and contributes to the development and advancement of the disease. Additionally, HDACi have been shown to enhance the cytotoxic effects of DNA-damaging agents such as radiation and cisplatin. In this study, we found that histone deacetylase inhibits valproic acid (VPA), synergized with PARP1 inhibitor (PARPi), talazoparib (BMN-673), and alkylating agent, and temozolomide (TMZ) to induce DNA damage and reduce glioblastoma multiforme. At the molecular level, VPA leads to a downregulation of FANCD2 and RAD51, and the eradication of glioblastoma cells. The results of this study indicate that combining HDACi with PARPi could potentially enhance the treatment of glioblastoma, the most aggressive type of cancer that originates in the brain. 10.3390/jpm13091315
FANCD2 Binding to H4K20me2 via a Methyl-Binding Domain Is Essential for Efficient DNA Cross-Link Repair. Paquin Karissa L,Mamrak Nicholas E,Garzon Jada L,Cantres-Velez Juan A,Azzinaro Paul A,Vuono Elizabeth A,Lima Kevin E,Camberg Jodi L,Howlett Niall G Molecular and cellular biology Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure and increased cancer risk. FA is caused by mutation of any 1 of 22 genes, and the FA proteins function cooperatively to repair DNA interstrand cross-links (ICLs). A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs within chromatin. How FANCD2 and FANCI are anchored to chromatin remains unknown. In this study, we identify and characterize a FANCD2 histone-binding domain (HBD) and embedded methyl-lysine-binding domain (MBD) and demonstrate binding specificity for H4K20me2. Disruption of the HBD/MBD compromises FANCD2 chromatin binding and nuclear focus formation and its ability to promote error-free DNA interstrand cross-link repair, leading to increased error-prone repair and genome instability. Our study functionally describes the first FA protein chromatin reader domain and establishes an important link between this human genetic disease and chromatin plasticity. 10.1128/MCB.00194-19
Regulation of the Fanconi Anemia DNA Repair Pathway by Phosphorylation and Monoubiquitination. Ishiai Masamichi Genes The Fanconi anemia (FA) DNA repair pathway coordinates a faithful repair mechanism for stalled DNA replication forks caused by factors such as DNA interstrand crosslinks (ICLs) or replication stress. An important role of FA pathway activation is initiated by monoubiquitination of FANCD2 and its binding partner of FANCI, which is regulated by the ATM-related kinase, ATR. Therefore, regulation of the FA pathway is a good example of the contribution of ATR to genome stability. In this short review, we summarize the knowledge accumulated over the years regarding how the FA pathway is activated via phosphorylation and monoubiquitination. 10.3390/genes12111763
Characterization of FANCL variants observed in patient cancer cells. Bioscience reports Fanconi Anemia (FA) is a rare genetic disorder characterized by developmental defects, bone marrow failure and high predisposition to cancer. The FA DNA repair pathway is required in humans to coordinate repair of DNA interstrand cross-links. The central event in the activation of the pathway is the monoubiquitination of FANCD2 and FANCI by the E2-E3 pair, Ube2T-FANCL, with the central UBC-RWD (URD) domain of FANCL recognizing the substrates. Whole genome sequencing studies of cancer cells from patients identified point mutations in the FANCL URD domain. We analysed 17 such variants of FANCL, including known substrate binding mutants (W212A, W214A and L248A, F252A, L254A, I265A), a FA mutation (R221C) and 14 cancer-associated mutations (F110S, I136V, L149V, L154S, A192G, E215Q, E217K, R221W, T224K, M247V, F252L, N270K, V287G, E289Q) through recombinant expression analysis, thermal shift assay, interaction with FANCD2, in vitro ubiquitination activity, and cellular sensitivity to an interstrand cross-linking agent. We find that the FANCL mutations I136V, L154S, W212A and L214A, R221W, R221C, and V287G are destabilizing, with N270K and E289Q destabilizing the C-terminal helices of the URD domain. The hydrophobic patch mutant (L248A, F252A, L254A, I265A), along with mutations E217K, T224K, and M247V, cause defects in the catalytic function of FANCL. This highlights the C-terminal lobe of the FANCL URD domain as important for the activity and function of FANCL. These mutations which affect the fold and activity of FANCL may contribute to tumorigenesis in these non-FA cancer patients, and this implicates FA genes in general cancer progression. 10.1042/BSR20191304
Fanconi anemia associated protein 20 (FAAP20) plays an essential role in homology-directed repair of DNA double-strand breaks. Communications biology FAAP20 is a Fanconi anemia (FA) protein that associates with the FA core complex to promote FANCD2/FANCI monoubiquitination and activate the damage response to interstrand crosslink damage. Here, we report that FAAP20 has a marked role in homologous recombination at a DNA double-strand break not associated with an ICL and separable from its binding partner FANCA. While FAAP20's role in homologous recombination is not dependent on FANCA, we found that FAAP20 stimulates FANCA's biochemical activity in vitro and participates in the single-strand annealing pathway of double-strand break repair in a FANCA-dependent manner. This indicates that FAAP20 has roles in several homology-directed repair pathways. Like other homology-directed repair factors, FAAP20 loss causes a reduction in nuclear RAD51 Irradiation-induced foci; and sensitizes cancer cells to ionizing radiation and PARP inhibition. In summary, FAAP20 participates in DNA double strand break repair by supporting homologous recombination in a non-redundant manner to FANCA, and single-strand annealing repair via FANCA-mediated strand annealing activity. 10.1038/s42003-023-05252-9
Allosteric mechanism for site-specific ubiquitination of FANCD2. Chaugule Viduth K,Arkinson Connor,Rennie Martin L,Kämäräinen Outi,Toth Rachel,Walden Helen Nature chemical biology DNA-damage repair is implemented by proteins that are coordinated by specialized molecular signals. One such signal in the Fanconi anemia (FA) pathway for the repair of DNA interstrand crosslinks is the site-specific monoubiquitination of FANCD2 and FANCI. The signal is mediated by a multiprotein FA core complex (FA-CC) however, the mechanics for precise ubiquitination remain elusive. We show that FANCL, the RING-bearing module in FA-CC, allosterically activates its cognate ubiqutin-conjugating enzyme E2 UBE2T to drive site-specific FANCD2 ubiquitination. Unlike typical RING E3 ligases, FANCL catalyzes ubiquitination by rewiring the intraresidue network of UBE2T to influence the active site. Consequently, a basic triad unique to UBE2T engages a structured acidic patch near the target lysine on FANCD2. This three-dimensional complementarity, between the E2 active site and substrate surface, induced by FANCL is central to site-specific monoubiquitination in the FA pathway. Furthermore, the allosteric network of UBE2T can be engineered to enhance FANCL-catalyzed FANCD2-FANCI di-monoubiquitination without compromising site specificity. 10.1038/s41589-019-0426-z
Conserved function of Drosophila Fancd2 monoubiquitination in response to double-strand DNA breaks. G3 (Bethesda, Md.) Fanconi anemia genes play key roles in metazoan DNA damage responses, and human FA mutations cause numerous disease phenotypes. In human cells, activating monoubiquitination of the Fanconi anemia protein Fancd2 occurs following diverse DNA damage stimuli. Monoubiquitinated Fancd2 forms nuclear foci to recruit additional repair factors. Fancd2 animal models to date have focused on molecular nulls or whole gene knockdown, leaving the specific in vivo role of monoubiquitination unclear. Using a point mutant in a conserved residue, we recently linked Drosophila Fancd2 monoubiquitination to a mitosis-specific DNA double-strand break response. In this context, we used CRISPR/Cas9 to generate the first animal model of an endogenous mutation in the conserved monoubiquitination site (fancd2K595R). Here, we expand upon our characterization of fancd2K595R. We also introduce and characterize additional Drosophila tools to study fancd2, including new mutant alleles and GFP-tagged rescue transgenes. Using these new reagents, we show the impact of Drosophila Fancd2 on organismal and cell viability, as well as on repair protein localization, in the presence or absence of double-strand breaks. These findings expand our understanding of Fanconi anemia gene function in vivo and provide useful reagents for DNA repair research. 10.1093/g3journal/jkac129
RAD18 opposes transcription-associated genome instability through FANCD2 recruitment. PLoS genetics DNA replication is a vulnerable time for genome stability maintenance. Intrinsic stressors, as well as oncogenic stress, can challenge replication by fostering conflicts with transcription and stabilizing DNA:RNA hybrids. RAD18 is an E3 ubiquitin ligase for PCNA that is involved in coordinating DNA damage tolerance pathways to preserve genome stability during replication. In this study, we show that RAD18 deficient cells have higher levels of transcription-replication conflicts and accumulate DNA:RNA hybrids that induce DNA double strand breaks and replication stress. We find that these effects are driven in part by failure to recruit the Fanconi Anemia protein FANCD2 at difficult to replicate and R-loop prone genomic sites. FANCD2 activation caused by splicing inhibition or aphidicolin treatment is critically dependent on RAD18 activity. Thus, we highlight a RAD18-dependent pathway promoting FANCD2-mediated suppression of R-loops and transcription-replication conflicts. 10.1371/journal.pgen.1010309
Subcellular localization of FANCD2 is associated with survival in ovarian carcinoma. Joshi Sonali,Campbell Shawn,Lim Jeong Y,McWeeney Shannon,Krieg Adam,Bean Yukie,Pejovic Nadja,Mhawech-Fauceglia Paulette,Pejovic Tanja Oncotarget OBJECTIVE:Ovarian cancer is a leading cause of death from gynecological cancers. Late diagnosis and resistance to therapy results in mortality and effective screening is required for early diagnosis and better treatments. Expression of the Fanconi Anemia complementation group D2 protein (FANCD2) is reduced in ovarian surface epithelial cells (OSE) in patients with ovarian cancer. FANCD2 has been studied for its role in DNA repair; however multiple studies have suggested that FANCD2 has a role outside the nucleus. We sought to determine whether subcellular localization of FANCD2 correlates with patient outcome in ovarian cancer. METHODS:We examined the subcellular localization of FANCD2 in primary OSE cells from consenting patients with ovarian cancer or a normal ovary. Ovarian tissue microarray was stained with anti-FANCD2 antibody by immunohistochemistry and the correlation of FANCD2 localization with patient outcomes was assessed. FANCD2 binding partners were identified by immunoprecipitation of cytoplasmic FANCD2. RESULTS:Nuclear and cytoplasmic localization of FANCD2 was observed in OSEs from both normal and ovarian cancer patients. Patients with cytoplasmic localization of FANCD2 (cFANCD2) experienced significantly longer median survival time (50 months), versus patients without cytoplasmic localization of FANCD2 (38 months; < 0.05). Cytoplasmic FANCD2 was found to bind proteins involved in the innate immune system, cellular response to heat stress, amyloid fiber formation and estrogen mediated signaling. CONCLUSIONS:Our results suggest that the presence of cytoplasmic FANCD2 modulates FANCD2 activity resulting in better survival outcome in ovarian cancer patients. 10.18632/oncotarget.27437
TGFβ pathway is required for viable gestation of Fanconi anemia embryos. PLoS genetics Overexpression of the TGFβ pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFβ promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFβ signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFβ pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFβ and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFβ-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFβ-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFβ-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis. 10.1371/journal.pgen.1010459
Cyclin-Dependent Kinase-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity. Cantres-Velez Juan A,Blaize Justin L,Vierra David A,Boisvert Rebecca A,Garzon Jada L,Piraino Benjamin,Tan Winnie,Deans Andrew J,Howlett Niall G Molecular and cellular biology Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs upon exposure to DNA-damaging agents and during the S phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a cyclin-dependent kinase (CDK) regulatory phosphosite (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by immunoblotting with an S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during the S phase. Furthermore, FA-D2 () patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity. 10.1128/MCB.00234-21
FANCL supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner. Biochimica et biophysica acta. Molecular basis of disease Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The FA proteins have functions in genome maintenance and in the cytoplasmic process of selective autophagy, beyond their canonical roles of repairing DNA interstrand cross-links. FA core complex proteins FANCC, FANCF, FANCL, FANCA, FANCD2, BRCA1 and BRCA2, which previously had no known direct functions outside the nucleus, have recently been implicated in mitophagy. Although mutations in FANCL account for only a very small number of cases in FA families, it plays a key role in the FA pathophysiology and might drive carcinogenesis. Here, we demonstrate that FANCL protein is present in mitochondria in the control and Oligomycin and Antimycin (OA)-treated cells and its ubiquitin ligase activity is not required for its localization to mitochondria. CRISPR/Cas9-mediated knockout of FANCL in HeLa cells overexpressing parkin results in increased sensitivity to mitochondrial stress and defective clearing of damaged mitochondria upon OA treatment. This defect was reversed by the reintroduction of either wild-type FANCL or FANCL(C307A), a mutant lacking ubiquitin ligase activity. To summarize, FANCL protects from mitochondrial stress and supports Parkin-mediated mitophagy in a ubiquitin ligase-independent manner. 10.1016/j.bbadis.2022.166453
Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas. Moes-Sosnowska Joanna,Rzepecka Iwona K,Chodzynska Joanna,Dansonka-Mieszkowska Agnieszka,Szafron Lukasz M,Balabas Aneta,Lotocka Renata,Sobiczewski Piotr,Kupryjanczyk Jolanta Cancer biology & therapy OBJECTIVE:DNA repair pathways are potential targets of molecular therapy in cancer patients. The FANCD2, BRIP1, BRCA1/2, and FANCF genes are involved in homologous recombination DNA repair, which implicates their possible role in cell response to DNA-damaging agents. We evaluated a clinical significance of pre-treatment expression of these genes at mRNA level in 99 primary, advanced-stage ovarian carcinomas from patients, who later received taxane-platinum (TP) or platinum-cyclophosphamide (PC) treatment. METHODS:Gene expression was determined with the use of Real-Time PCR. The BRCA2 and BRIP1 gene sequence was investigated with the use of SSCP, dHPLC, and PCR-sequencing. RESULTS:Increased FANCD2 expression occurred to be a negative prognostic factor for all patients (PC+TP:HR 3.85, p = 0.0003 for the risk of recurrence; HR 1.96, p = 0.02 for the risk of death), and this association was even stronger in the TP-treated group (HR 6.7, p = 0.0002 and HR 2.33, p = 0.01, respectively). Elevated BRIP1 expression was the only unfavorable molecular factor in the PC-treated patients (HR 8.37, p = 0.02 for the risk of recurrence). Additionally, an increased FANCD2 and BRCA1/2 expression levels were associated with poor ovarian cancer outcome in either TP53-positive or -negative subgroups of the TP-treated patients, however these groups were small. Sequence analysis identified one protein truncating variant (1/99) in BRCA2 and no mutations (0/56) in BRIP1. CONCLUSIONS:Our study shows for the first time that FANCD2 overexpression is a strong negative prognostic factor in ovarian cancer, particularly in patients treated with TP regimen. Moreover, increased mRNA level of the BRIP1 is a negative prognostic factor in the PC-treated patients. Next, changes in the BRCA2 and BRIP1 genes are rare and together with other analyzed FA genes considered as homologous recombination deficiency may not affect the expression level of analyzed genes. 10.1080/15384047.2019.1579955
The Fanconi anemia core complex promotes CtIP-dependent end-resection to drive homologous recombination at DNA double-strand breaks. bioRxiv : the preprint server for biology Homologous Recombination (HR) is a high-fidelity repair mechanism of DNA Double-Strand Breaks (DSBs), which are induced by irradiation, genotoxic chemicals or physiological DNA damaging processes. DSBs are also generated as intermediates during the repair of interstrand crosslinks (ICLs). In this context, the Fanconi anemia (FA) core complex, which is effectively recruited to ICLs, promotes HR-mediated DSB-repair. However, whether the FA core complex also promotes HR at ICL-independent DSBs remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen with cells carrying the DSB-repair reporter DSB-Spectrum. Using isogenic cell-line models, we validated the HR-function of FANCL and Ube2T, and demonstrated a similar function for their ubiquitination-substrate FANCD2. We further show that FANCL and Ube2T are directly recruited to DSBs and are required for the accumulation of FANCD2 at these break sites. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of the nuclease CtIP at DSBs, and consequently for optimal end-resection and Rad51 loading. CtIP overexpression rescues HR in FANCL-deficient cells, validating that FANCL primarily regulates HR by promoting CtIP recruitment. Together, these data demonstrate that the FA core complex and FANCD2 have a dual genome maintenance function by promoting repair of DSBs as well as the repair of ICLs. 10.1101/2023.09.05.556391
FANCD2 modulates the mitochondrial stress response to prevent common fragile site instability. Fernandes Philippe,Miotto Benoit,Saint-Ruf Claude,Said Maha,Barra Viviana,Nähse Viola,Ravera Silvia,Cappelli Enrico,Naim Valeria Communications biology Common fragile sites (CFSs) are genomic regions frequently involved in cancer-associated rearrangements. Most CFSs lie within large genes, and their instability involves transcription- and replication-dependent mechanisms. Here, we uncover a role for the mitochondrial stress response pathway in the regulation of CFS stability in human cells. We show that FANCD2, a master regulator of CFS stability, dampens the activation of the mitochondrial stress response and prevents mitochondrial dysfunction. Genetic or pharmacological activation of mitochondrial stress signaling induces CFS gene expression and concomitant relocalization to CFSs of FANCD2. FANCD2 attenuates CFS gene transcription and promotes CFS gene stability. Mechanistically, we demonstrate that the mitochondrial stress-dependent induction of CFS genes is mediated by ubiquitin-like protein 5 (UBL5), and that a UBL5-FANCD2 dependent axis regulates the mitochondrial UPR in human cells. We propose that FANCD2 coordinates nuclear and mitochondrial activities to prevent genome instability. 10.1038/s42003-021-01647-8
Differential Regulation of Retinoic Acid Metabolism in Fanconi Anemia. bioRxiv : the preprint server for biology Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital abnormalities and increased risk for bone marrow failure and cancer. FA is caused by mutation of any one of 23 genes, the protein products of which function primarily in the maintenance of genome stability. An important role for the FA proteins in the repair of DNA interstrand crosslinks (ICLs) has been established . While the endogenous sources of ICLs relevant to the pathophysiology of FA have yet to be clearly determined, a role for the FA proteins in a two-tier system for the detoxification of reactive metabolic aldehydes has been established. To discover new metabolic pathways linked to FA, we performed RNA-seq analysis on non-transformed FA-D2 ( ) and FANCD2-complemented patient cells. Multiple genes associated with retinoic acid metabolism and signaling were differentially expressed in FA-D2 ( ) patient cells, including and , which encode for retinaldehyde and retinol dehydrogenases, respectively. Increased levels of the ALDH1A1 and RDH10 proteins was confirmed by immunoblotting. FA-D2 ( ) patient cells displayed increased aldehyde dehydrogenase activity compared to the FANCD2-complemented cells. Upon exposure to retinaldehyde, FA-D2 ( ) cells exhibited increased DNA double-strand breaks and checkpoint activation indicative of a defect in the repair of retinaldehyde-induced DNA damage. Our findings describe a novel link between retinoic acid metabolism and FA and identify retinaldehyde as an additional reactive metabolic aldehyde relevant to the pathophysiology of FA. 10.1101/2023.04.06.535759
TFG-maintaining stability of overlooked FANCD2 confers early DNA-damage response. Ma Chi,Hokutan Kanani,Shen Yihang,Nepal Manoj,Kim Jin-Hee,Zhang Jun,Fei Peiwen Aging Emerging Fanconi Anemia (FA) signaling in the field of cancer research annotates the extreme importance of its center player, Fanconi Anemia complementation group D2 (FANCD2) in protecting human cells from going awry. However, a previously-unrecognized form of FANCD2, namely FANCD2-V2, is understudied. We report TRK-Fused Gene (TFG) is critical for roles played by FANCD2-V2 in early responses to DNA damage, but not for FANCD2-V1, the long-known form of FANCD2. FANCD2-V2 forms nuclear foci upon DNA damage, and both its focus appearance and disappearance are earlier than FANCD2-V1. The amino acid/aa 5-100 of TFG and the aa1437-1442 of FANCD2-V2 were identified to contribute to their interaction, which maintains the steady-state level of FANCD2-V2 protein. TFGΔaa5-100 or FANCD2-V2Δaa1437-1442-carrying cells could not show timely focus formation of FANCD2-V2 upon DNA damage and gained carcinogenicity over time. This study provides a previously-unknown key to unlock in-depth insights into maintaining genome stability, fostering translational studies on preventing, diagnosing and/or treating related diseases. 10.18632/aging.103782
Inhibition of USP1 enhances anticancer drugs-induced cancer cell death through downregulation of survivin and miR-216a-5p-mediated upregulation of DR5. Cell death & disease Ubiquitin-specific protease 1 (USP1) is a deubiquitinase involved in DNA damage repair by modulating the ubiquitination of major regulators, such as PCNA and FANCD2. Because USP1 is highly expressed in many cancers, dysregulation of USP1 contributes to cancer therapy. However, the role of USP1 and the mechanisms underlying chemotherapy remain unclear. In this study, we found high USP1 expression in tumor tissues and that it correlated with poor prognosis in RCC. Mechanistically, USP1 enhanced survivin stabilization by removing ubiquitin. Pharmacological inhibitors (ML23 and pimozide) and siRNA targeting USP1 induced downregulation of survivin expression. In addition, ML323 upregulated DR5 expression by decreasing miR-216a-5p expression at the post-transcriptional level, and miR-216a-5p mimics suppressed the upregulation of DR5 by ML323. Inhibition of USP1 sensitized cancer cells. Overexpression of survivin or knockdown of DR5 markedly prevented the co-treatment with ML323 and TRAIL-induced apoptosis. These results of in vitro were proved in a mouse xenograft model, in which combined treatment significantly reduced tumor size and induced survivin downregulation and DR5 upregulation. Furthermore, USP1 and survivin protein expression showed a positive correlation, whereas miR-216a-5p and DR5 were inversely correlated in RCC tumor tissues. Taken together, our results suggest two target substrates of USP1 and demonstrate the involvement of survivin and DR5 in USP1-targeted chemotherapy. 10.1038/s41419-022-05271-0
Safeguarding DNA Replication: A Golden Touch of MiDAS and Other Mechanisms. International journal of molecular sciences DNA replication is a tightly regulated fundamental process allowing the correct duplication and transfer of the genetic information from the parental cell to the progeny. It involves the coordinated assembly of several proteins and protein complexes resulting in replication fork licensing, firing and progression. However, the DNA replication pathway is strewn with hurdles that affect replication fork progression during S phase. As a result, cells have adapted several mechanisms ensuring replication completion before entry into mitosis and segregating chromosomes with minimal, if any, abnormalities. In this review, we describe the possible obstacles that a replication fork might encounter and how the cell manages to protect DNA replication from S to the next G1. 10.3390/ijms231911331
The RPA inhibitor HAMNO sensitizes Fanconi anemia pathway-deficient cells. Cell cycle (Georgetown, Tex.) The Fanconi anemia (FA) DNA repair pathway is required for DNA inter-strand crosslink (ICL) repair. Besides its role in ICL repair, FA proteins play a central role in stabilizing stalled replication forks, thereby ensuring genome integrity. We previously demonstrated that depletion of replication protein A (RPA) induces the activation of FA pathway leading to FANCD2 monoubiquitination and FANCD2 foci formation. Thus, we speculated that FA-deficient cells would be more sensitive to RPA inhibition compared to FA-proficient cells. Following treatment with RPA inhibitor HAMNO, we observed significant induction in FANCD2 monoubiquitination and foci formation as observed in RPA depletion. In addition, HAMNO treatment caused increased levels of γ-H2AX and S-phase accumulation in FA-deficient cells. Importantly, FA-deficient cells showed more increased sensitivity to HAMNO than FA-proficient cells. Moreover, in combination with cisplatin, HAMNO further enhanced the cytotoxicity of cisplatin in FA-deficient cells, while being less toxic against FA-proficient cells. This result suggests that RPA inhibition might be a potential therapeutic candidate for the treatment of FA pathway-deficient tumors. 10.1080/15384101.2022.2074200
Integrative analyses of prognosis, tumor immunity, and ceRNA network of the ferroptosis-associated gene FANCD2 in hepatocellular carcinoma. Frontiers in genetics Extensive evidence has revealed that ferroptosis plays a vital role in HCC development and progression. Fanconi anemia complementation group D2 (FANCD2) has been reported to serve as a ferroptosis-associated gene and has a close relationship with tumorigenesis and drug resistance. However, the impact of the FANCD2-related immune response and its mechanisms in HCC remains incompletely understood. In the current research, we evaluated the prognostic significance and immune-associated mechanism of FANCD2 based on multiple bioinformatics methods and databases. The results demonstrated that FANCD2 was commonly upregulated in 15/33 tumors, and only the high expression of FANCD2 in HCC was closely correlated with worse clinical outcomes by OS and DFS analyses. Moreover, ncRNAs, including two major types, miRNAs and lncRNAs, were closely involved in mediating FANCD2 upregulation in HCC and were established in a ceRNA network by performing various analyses. The DUXAP8-miR-29c-FANCD2 and LINC00511-miR-29c-FANCD2 axes were identified as the most likely ncRNA-associated upstream regulatory axis of FANCD2 in HCC. Finally, FANCD2 expression was confirmed to be positively related to HCC immune cell infiltration, immune checkpoints, and IPS analysis, and GSEA results also revealed that this ferroptosis-associated gene was primarily involved in cancer-associated pathways in HCC. In conclusion, our investigations indicate that ncRNA-related modulatory overexpression of FANCD2 might act as a promising prognostic and immunotherapeutic target against HCC. 10.3389/fgene.2022.955225
FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair. Nature structural & molecular biology Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2-FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors. 10.1038/s41594-020-0380-1
Severe Fanconi Anemia phenotypes in Fancd2 depletion mice. Yang Qiao,Xie Hui,Zhong Yixinhe,Li Dongbo,Ke Xianfu,Ying Huazhong,Yu Bing,Zhang Tingting Biochemical and biophysical research communications Fanconi anemia (FA) is a genetic disorder characterized by congenital malfunction, bone marrow failure and hypersensitivity to DNA damage. FANCD2 protein play the central role in FA pathway. To study the in vivo role of FANCD2, we generated and characterized a new Fancd2 knockout mouse strain with 7bp deletion in Fancd2 gene 5' terminus using Crispr-Cas9 in congenic C57BL/6J background. This Fancd2 mice displayed similar but overall more severe manifestation than the previous ES cell targeted Fancd2 model. These features include increased embryonic and postnatal lethality rate, higher incidence of microphthalmia, and more severe hypogonadism. The anemia we observed in this Fancd2 mice has not been described in other FA models. Further study indicated that the hematopoiesis deficiency was associated with increased apoptotic cell death, G2/M phase arrest and hypersensitivity to MMC and IR damage of Fancd2 bone marrow progenitor cells. Collectively, the resulting Fancd2 mice with higher resemblance of FA patient symptoms, will be useful in understand the parthenogenesis of pancytopenia and bone marrow failure in FA. 10.1016/j.bbrc.2019.04.201
Research progress of the Fanconi anemia pathway and premature ovarian insufficiency†. Biology of reproduction The Fanconi anemia pathway is a key pathway involved in the repair of deoxyribonucleic acidinterstrand crosslinking damage, which chiefly includes the following four modules: lesion recognition, Fanconi anemia core complex recruitment, FANCD2-FANCI complex monoubiquitination, and downstream events (nucleolytic incision, translesion synthesis, and homologous recombination). Mutations or deletions of multiple Fanconi anemia genes in this pathway can damage the interstrand crosslinking repair pathway and disrupt primordial germ cell development and oocyte meiosis, thereby leading to abnormal follicular development. Premature ovarian insufficiency is a gynecological clinical syndrome characterized by amenorrhea and decreased fertility due to decreased oocyte pool, accelerated follicle atresia, and loss of ovarian function in women <40 years old. Furthermore, in recent years, several studies have detected mutations in the Fanconi anemia gene in patients with premature ovarian insufficiency. In addition, some patients with Fanconi anemia exhibit symptoms of premature ovarian insufficiency and infertility. The Fanconi anemia pathway and premature ovarian insufficiency are closely associated. 10.1093/biolre/ioad110
Structural basis of FANCD2 deubiquitination by USP1-UAF1. Nature structural & molecular biology Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1-UAF1 complex is the monoubiquitinated FANCI-FANCD2 heterodimer, which is involved in the repair of DNA interstrand crosslinks via the Fanconi anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin and bound to monoubiquitinated FANCI-FANCD2. The crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1, two related proteases. A cryo-EM reconstruction of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition. 10.1038/s41594-021-00576-8
ATR-Mediated FANCI Phosphorylation Regulates Both Ubiquitination and Deubiquitination of FANCD2. Frontiers in cell and developmental biology DNA interstrand crosslinks (ICLs) are a physical barrier to replication and therefore toxic to cell viability. An important mechanism for the removal of ICLs is the Fanconi Anemia DNA repair pathway, which is initiated by mono-ubiquitination of FANCD2 and its partner protein FANCI. Here, we show that maintenance of FANCD2 and FANCI proteins in a monoubiquitinated form is regulated by the ATR-kinase. Using recombinant proteins in biochemical reconstitution experiments we show that ATR directly phosphorylates FANCI on serine 556, 559, and 565 to stabilize its association with DNA and FANCD2. This increased association with DNA stimulates the conjugation of ubiquitin to both FANCI and FANCD2, but also inhibits ubiquitin deconjugation. Using phosphomimetic and phosphodead mutants of FANCI we show that S559 and S565 are particularly important for protecting the complex from the activity of the deubiquitinating enzyme USP1:UAF1. Our results reveal a major mechanism by which ATR kinase maintains the activation of the FA pathway, by promoting the accumulation of FANCD2 in the ubiquitinated form active in DNA repair. 10.3389/fcell.2020.00002
FANCD2-dependent mitotic DNA synthesis relies on PCNA K164 ubiquitination. Cell reports Ubiquitination of proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) activates DNA damage tolerance pathways. Currently, we lack a comprehensive understanding of how PCNA K164 ubiquitination promotes genome stability. To evaluate this, we generated stable cell lines expressing PCNA from the endogenous PCNA locus. Our data reveal that the inability to ubiquitinate K164 causes perturbations in global DNA replication. Persistent replication stress generates under-replicated regions and is exacerbated by the DNA polymerase inhibitor aphidicolin. We show that these phenotypes are due, in part, to impaired Fanconi anemia group D2 protein (FANCD2)-dependent mitotic DNA synthesis (MiDAS) in PCNA cells. FANCD2 mono-ubiquitination is significantly reduced in PCNA mutants, leading to reduced chromatin association and foci formation, both prerequisites for FANCD2-dependent MiDAS. Furthermore, K164 ubiquitination coordinates direct PCNA/FANCD2 colocalization in mitotic nuclei. Here, we show that PCNA K164 ubiquitination maintains human genome stability by promoting FANCD2-dependent MiDAS to prevent the accumulation of under-replicated DNA. 10.1016/j.celrep.2023.113523
Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination. Liang Zhuobin,Liang Fengshan,Teng Yaqun,Chen Xiaoyong,Liu Jingchun,Longerich Simonne,Rao Timsi,Green Allison M,Collins Natalie B,Xiong Yong,Lan Li,Sung Patrick,Kupfer Gary M Cell reports Fanconi anemia (FA) is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. FA cells are hypersensitive to DNA replicative stress and accumulate co-transcriptional R-loops. Here, we use the Damage At RNA Transcription assay to reveal colocalization of FANCD2 with R-loops in a highly transcribed genomic locus upon DNA damage. We further demonstrate that highly purified human FANCI-FANCD2 (ID2) complex binds synthetic single-stranded RNA (ssRNA) and R-loop substrates with high affinity, preferring guanine-rich sequences. Importantly, we elucidate that human ID2 binds an R-loop structure via recognition of the displaced ssDNA and ssRNA but not the RNA:DNA hybrids. Finally, a series of RNA and R-loop substrates are found to strongly stimulate ID2 monoubiquitination, with activity corresponding to their binding affinity. In summary, our results support a mechanism whereby the ID2 complex suppresses the formation of pathogenic R-loops by binding ssRNA and ssDNA species, thereby activating the FA pathway. 10.1016/j.celrep.2018.12.084
DNA repair protein FANCD2 has both ubiquitination-dependent and ubiquitination-independent functions during germ cell development. The Journal of biological chemistry When DNA interstrand crosslink lesions occur, a core complex of Fanconi anemia proteins promotes the ubiquitination of FANCD2 and FANCI, which recruit downstream factors to repair the lesion. However, FANCD2 maintains genome stability not only through its ubiquitination-dependent but also its ubiquitination-independent functions in various DNA damage response pathways. Increasing evidence suggests that FANCD2 is essential for fertility, but its ubiquitination-dependent and ubiquitination-independent roles during germ cell development are not well characterized. In this study, we analyzed germ cell development in Fancd2 KO and ubiquitination-deficient mutant (Fancd2) mice. We showed that in the embryonic stage, both the ubiquitination-dependent and ubiquitination-independent functions of FANCD2 were required for the expansion of primordial germ cells and establishment of the reproductive reserve by reducing transcription-replication conflicts and thus maintaining genome stability in primordial germ cells. Furthermore, we found that during meiosis in spermatogenesis, FANCD2 promoted chromosome synapsis and regulated crossover formation independently of its ubiquitination, but that both ubiquitinated and nonubiquitinated FANCD2 functioned in programmed double strand break repair. Finally, we revealed that on meiotic XY chromosomes, H3K4me2 accumulation required ubiquitination-independent functionality of FANCD2, while the regulation of H3K9me2 and H3K9me3 depended on FANCD2 ubiquitination. Taken together, our findings suggest that FANCD2 has distinct functions that are both dependent on and independent of its ubiquitination during germ cell development. 10.1016/j.jbc.2023.102905
Regulation of R-loops and genome instability in Fanconi anemia. Okamoto Yusuke,Hejna James,Takata Minoru Journal of biochemistry Fanconi anemia (FA) is a devastating hereditary disorder with impaired genome stability resulting in physical abnormalities, gradual loss of hematopoietic stem cells and development of tumours and leukaemia. It has been suggested that functions of FA genes are required to maintain genome stability by counteracting endogenous metabolites, such as aldehydes, that damage DNA and stall replication forks. Recent studies have implicated co-transcriptional R-loops, consisting of a DNA:RNA hybrid and displaced single-stranded DNA, as one of the potential endogenous sources that induce genome instability and the FA phenotype. This review focuses on recent literature, including our own, regarding the interplay between FA proteins and R-loops, and will provide readers with a concise summary of this rapidly evolving field. 10.1093/jb/mvz019
Abnormally Expressed Ferroptosis-Associated FANCD2 in Mediating the Temozolomide Resistance and Immune Response in Glioblastoma. Frontiers in pharmacology Ferroptosis-related genes (FRGs) have been identified as potential targets involved in oncogenesis and cancer therapeutic response. Nevertheless, the specific roles and underlying mechanisms of FRGs in GBM and temozolomide (TMZ) resistance remain unclear. Through comprehensive bioinformatics, we found that ferroptosis-related Fanconi anemia complementation group D2 (FANCD2) was significantly up-regulated in GBM tissues, and the high expression level of FANCD2 was related to the poor prognosis in primary and recurrent GBM patients. Furthermore, FANCD2 could promote TMZ resistance by attenuating ferroptosis in GBM cells. Knockdown of FANCD2 could increase reactive oxygen species (ROS) levels and inhibit cell survival. The two characteristics were associated with ferroptosis in TMZ-resistant GBM cells T98G-R and U118-R. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that aberrantly expressed FANCD2 was potentially linked with several cancer-associated signaling pathways, including chromosome segregation, DNA replication, and cell cycle transition. In addition, we demonstrated that FANCD2 expression was positively correlated with several tumor-infiltrating lymphocytes (TILs) and multiple immune-associated signatures in GBM. Therefore, up-regulated FANCD2 could protect GBM cells from ferroptosis and promote TMZ resistance. FANCD2 may be a novel therapeutic target in GBM. 10.3389/fphar.2022.921963
Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins. Chaugule Viduth K,Arkinson Connor,Toth Rachel,Walden Helen Methods in enzymology In higher eukaryotes, DNA damage repair response pathways are orchestrated by several molecular signals including ubiquitination. In particular the repair of DNA interstrand crosslinks, toxic to transcription and replication processes, involve the activation of the Fanconi anemia repair pathway. At the heart of this pathway lies the monoubiquitination of FANCD2 and FANCI proteins, which triggers the recruitment of DNA repair factors. A major road block in our understanding of this fundamental repair pathway arises from the challenge with generating sufficient quantities of site-specifically monoubiquitinated FANCD2 and FANCI proteins to enable mechanistic and molecular studies. Current in vitro methods rely on the purification of a large (~0.8MDa), multiprotein E3 complex that can only partially monoubiquitinate a FANCD2-FANCI-DNA complex. In this chapter, we describe detailed protocols for the preparation of homogeneously and natively monoubiquitinated FANCD2 and FANCI proteins in isolation. The method relies on the use of a minimal E3 module and an engineered E2 variant that together drive site-specific ubiquitination of the isolated substrates, without the requirement of DNA cofactors. Using the enzymatic approach, we also demonstrate how added functionalities such as a fluorescently labeled ubiquitin can be conjugated on the FANCD2 and FANCI substrates, thus enabling multiple downstream applications. 10.1016/bs.mie.2018.12.021
Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage. Cell reports Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway. 10.1016/j.celrep.2019.05.003
Optimized structure of monoubiquitinated FANCD2 (human) at Lys 561: a theoretical approach. Journal of biomolecular structure & dynamics Fanconi anaemia pathway repairs inter-strand cross linking damage (ICL) of the DNA. Monoubiquitination of FANCD2 and FANCI is very crucial for ICL repairing. In this work we have tried to understand the monoubiquitinated FANCD2 structure, which facilitates the FANCD2 for binding the damage part of the chromatin. Crystal structure of the monoubiquitinated FANCD2 alone is not available, therefore we have modelled the optimized structure of the human monoubiquitinated (Lys 561) FANCD2. As there is no suitable software or web server we have developed a method for building up monoubiquitinated product and validated on simplest monoubiquitinated protein, diubiquitin. We have predicted the structure of human monoubiquitinated FANCD2 by using our method and studied the interaction with DNA by docking studies. Molecular Dynamics (MD) simulation has been used to understand the stability of the structure. Large structural differences have been observed between FANCD2 and monoubiquitinated FANCD2. Docking studies with DNA suggest that the binding site varies for the FANCD2 and monoubiquitinated FANCD2.Communicated by Ramaswamy H. Sarma. 10.1080/07391102.2021.1929490
A Fanci knockout mouse model reveals common and distinct functions for FANCI and FANCD2. Dubois Emilie L,Guitton-Sert Laure,Béliveau Mariline,Parmar Kalindi,Chagraoui Jalila,Vignard Julien,Pauty Joris,Caron Marie-Christine,Coulombe Yan,Buisson Rémi,Jacquet Karine,Gamblin Clémence,Gao Yuandi,Laprise Patrick,Lebel Michel,Sauvageau Guy,D d'Andrea Alan,Masson Jean-Yves Nucleic acids research Fanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci -/- mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci-/- Fancd2-/- also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis. 10.1093/nar/gkz514
Ferroptosis regulator FANCD2 is associated with immune infiltration and predicts worse prognosis in lung adenocarcinoma. Frontiers in genetics Lung adenocarcinoma (LUAD) remains one of the leading causes of cancer-related death. Although immunotherapy has been shown to improve survival in LUAD patients, only a select group of LUAD patients could benefit from it. The correlation between ferroptosis and the tumor immune environment requires further investigation in the setting of LUAD. An analysis using The Cancer Genome Atlas (TCGA)-LUAD cohort systematically evaluated the expression levels of ferroptosis regulators between LUAD and normal tissues and demonstrated the correlation of ferroptosis regulators with the immune checkpoint B7-H3 expression. Based on consensus clustering analysis, we divided LUAD patients into two subtypes according to the expression pattern of ferroptosis regulators. Cluster 2 patients showed more favorable overall survival (OS) ( < 0.001) and disease-free survival (DFS) ( < 0.001) than Cluster 1 patients. CIBERSORT analysis indicated that Cluster 1 patients harbored higher infiltrated levels of uncharacterized cells, CD4 T cells (nonregulatory), and myeloid dendritic cells, while Cluster 2 patients were more correlated with B cells, M1 macrophages, natural killer cells (NK cells) and regulatory T cells (Tregs). More importantly, we identified FANCD2 as a potentially unfavorable prognostic factor that was overexpressed in LUAD and positively associated with the checkpoint molecule B7-H3 expression. In addition, higher FANCD2 expression was related to a higher tumor immune dysfunction and exclusion (TIDE) score, indicating lower responder rates to cancer immunotherapeutics. In summary, our study suggested a relationship between immune infiltration and ferroptosis and that FANCD2 is a potential biomarker for clinical outcomes and a therapeutic target for LUAD therapy concerning ferroptotic regulation. Our findings may help to advance personalized treatment and improve the prognosis of LUAD. 10.3389/fgene.2022.922914
Loss of FANCD2 and related proteins may predict malignant transformation in oral epithelial dysplasia. Oral surgery, oral medicine, oral pathology and oral radiology OBJECTIVE:Predicting malignant transformation (MT) in oral epithelial dysplasia (OED) is challenging. The higher rate of MT reported in nonsmokers suggests an endogenous etiology in oncogenesis. We hypothesize that loss of FANCD2 and associated proteins could influence genomic instability and MT in the absence of environmental carcinogens. STUDY DESIGN:Longitudinal archival samples were obtained from 40 individuals with OED: from diagnosis to the most recent review in 23 patients with stable OED or until excision of the squamous cell carcinoma in 17 patients with unstable OED undergoing MT. Histopathological reassessment, immunohistochemistry for FANCD2, and Western blotting for phosphorylation/monoubiquitylation status of ATR, CHK1, FANCD2, and FANCG were undertaken on each tissue sample. RESULTS:Decreased expression of FANCD2 was observed in the diagnostic biopsies of OED lesions that later underwent MT. Combining the FANCD2 expression scores with histologic grading more accurately predicted MT (P = .005) than histology alone, and it correctly predicted MT in 10 of 17 initial biopsies. Significantly reduced expression of total FANCD2, pFANCD2, pATR, pCHK-1, and pFANCG was observed in unstable OED. CONCLUSIONS:There is preliminary evidence that defects in the DNA damage sensing/signaling/repair cascade are associated with MT in OED. Loss of expression of FANCD2 protein in association with a higher histologic grade of dysplasia offered better prediction of MT than clinicopathologic parameters alone. 10.1016/j.oooo.2021.07.001
The Interplay Between the DNA Damage Response, RNA Processing and Extracellular Vesicles. Meng Xiangbing,Yang Shujie,Camp Vanessa J A Frontiers in oncology RNA processing was recently found to affect DNA damage response. The RNA processing factors THRAP3 and BCLAF1 play critical role in keeping DNA genomic stability by regulating the transcription, mRNA splicing and export of DNA repair proteins BRCA2, PALB2, Rad51, FANCD2, and FANCL in response to DNA damage. RNA processing factors THRAP3 and BCLAF1 play critical roles in maintaining DNA genomic stability. These factors regulate transcription, mRNA splicing and nuclear RNA export of DNA repair proteins BRCA2, PALB2, Rad51, FANCD2, and FANCL in response to DNA damage. Splicing factors SRSF10 and Sam68 were found to control the DNA damage agent-induced mRNA splicing of transcripts including BCLAF1, BRCA1, BCL2L1, CASP8, CHK2, and RBBP8 to regulate apoptosis, cell-cycle transition and DNA repair. Splicing factors and RNA binding proteins (RBPs) were also found to play a critical role in DNA/RNA hybrids (R-loops) formed during transcription and RNA processing to prevent RNA-induced genome instability. At the same time, DNA repair proteins FANCI and FANCD2 were found to regulate the nuclear localization of splicing factors SF3B1 in the DNA damage response. In addition, tumor-derived extracellular vesicles (Evs) enhanced by chemotherapeutic agents in cancer were found to promote cancer metastasis and drug resistance. Inhibiting Evs from cancer cells significantly reduced cancer metastasis and drug resistance. Furthermore, cross-talk between the DNA damage response and the immune response was observed including the enhancement of the efficacy of immune checkpoint blockade by PARP inhibitors and the effect of PD-L1 on mRNA stability of various mRNAs involved in DNA damage response by acting as a novel RNA binding protein to increase drug resistance in cancer cells. This review will introduce recent progress on the interplay of the DNA damage response, the RNA processing and the extracellular vesicles mediated metastasis. 10.3389/fonc.2019.01538
Fancd2-deficient hematopoietic stem and progenitor cells depend on augmented mitochondrial translation for survival and proliferation. Chatla Srinivas,Du Wei,Wilson Andrew F,Meetei Amom Ruhikanta,Pang Qishen Stem cell research Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes including response to DNA damage and oxidative stress. Here we show that a major FA protein, Fancd2, plays a role in mitochondrial biosynthesis through regulation of mitochondrial translation. Fancd2 interacts with Atad3 and Tufm, which are among the most frequently identified components of the mitochondrial nucleoid complex essential for mitochondrion biosynthesis. Deletion of Fancd2 in mouse hematopoietic stem and progenitor cells (HSPCs) leads to increase in mitochondrial number, and enzyme activity of mitochondrion-encoded respiratory complexes. Fancd2 deficiency increases mitochondrial protein synthesis and induces mitonuclear protein imbalance. Furthermore, Fancd2-deficient HSPCs show increased mitochondrial respiration and mitochondrial reactive oxygen species. By using a cell-free assay with mitochondria isolated from WT and Fancd2-KO HSPCs, we demonstrate that the increased mitochondrial protein synthesis observed in Fancd2-KO HSPCs was directly linked to augmented mitochondrial translation. Finally, Fancd2-deficient HSPCs are selectively sensitive to mitochondrial translation inhibition and depend on augmented mitochondrial translation for survival and proliferation. Collectively, these results suggest that Fancd2 restricts mitochondrial activity through regulation of mitochondrial translation, and that augmented mitochondrial translation and mitochondrial respiration may contribute to HSC defect and bone marrow failure in FA. 10.1016/j.scr.2019.101550
FANCM regulates repair pathway choice at stalled replication forks. Molecular cell Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer. 10.1016/j.molcel.2021.03.044
FANCD2 as a novel prognostic biomarker correlated with immune and drug therapy in Hepatitis B-related hepatocellular carcinoma. European journal of medical research BACKGROUND:Ferroptosis is related to the immunosuppression of tumors and plays a critical role in cancer progression. Fanconi anemia complementation group D2 (FANCD2) is a vital gene that regulates ferroptosis. However, the mechanism of action of FANCD2 in Hepatitis B-related hepatocellular carcinoma (HCC) remains unknown. In this study, we investigated the prognostic significance and mechanism of action of FANCD2 in Hepatitis B-related HCC. METHODS:The expression of FANCD2 in Hepatitis B-related HCC was explored using The Cancer Genome Atlas (TCGA) and validated using the Gene Expression Omnibus (GEO) database. Univariate and multivariate Cox regression analyses and Kaplan-Meier survival curves were used to analyze the relationship between FANCD2 expression and the overall survival of patients with Hepatitis B-related HCC. Protein-protein interaction networks for FANCD2 were built using the STRING website. In addition, correlations between FANCD2 expression and the dryness index, tumor mutational burden, microsatellite instability (MSI), immune pathways, genes involved in iron metabolism, and sorafenib chemotherapeutic response were analyzed. RESULTS:Our results indicated that FANCD2 was significantly overexpressed in Hepatitis B-related HCC and demonstrated a strong predictive ability for diagnosis (Area Under Curve, 0.903) and prognosis of the disease. High FANCD2 expression was associated with poor prognosis, high-grade tumors, high expression of PDL-1, high MSI scores, and low sorafenib IC50 in Hepatitis B-related HCC. BRCA1, BRCA2, FAN1, and FANCC were vital proteins interacting with FANCD2. The expression level of FANCD2 significantly correlated with the infiltration levels of Treg cells, B cells, CD8 + T cells, CD4 + T cells, neutrophils, macrophages, myeloid dendritic cells, and NK cells in Hepatitis B-related HCC. FANCD2 was positively correlated with the tumor proliferation signature pathway, DNA repair, and cellular response to hypoxia. CONCLUSION:Our study indicated that FANCD2 was a potential novel biomarker and immunotherapeutic target against Hepatitis B-related HCC, which might be related to the chemotherapeutic response to sorafenib. 10.1186/s40001-023-01411-0
Regulation of Replication Stress in Alternative Lengthening of Telomeres by Fanconi Anaemia Protein. Genes Fanconi anaemia (FA)-related proteins function in interstrand crosslink (ICL) repair pathways and multiple damage repair pathways. Recent studies have found that FA proteins are involved in the regulation of replication stress (RS) in alternative lengthening of telomeres (ALT). Since ALT cells often exhibit high-frequency ATRX mutations and high levels of telomeric secondary structure, high levels of DNA damage and replicative stress exist in ALT cells. Persistent replication stress is required to maintain the activity of ALT mechanistically, while excessive replication stress causes ALT cell death. FA proteins such as FANCD2 and FANCM are involved in the regulation of this balance by resolving or inhibiting the formation of telomere secondary structures to stabilize stalled replication forks and promote break-induced repair (BIR) to maintain the survival of ALT tumour cells. Therefore, we review the role of FA proteins in replication stress in ALT cells, providing a rationale and direction for the targeted treatment of ALT tumours. 10.3390/genes13020180
FANCD2 promotes the malignant behavior of endometrial cancer cells and its prognostic value. Experimental cell research Defective DNA damage repair is a key mechanism affecting tumor susceptibility, treatment response, and survival outcome of endometrial cancer (EC). Fanconi anemia complementation group D2 (FANCD2) is the core component of the Fanconi anemia repair pathway. To explore the function of FANCD2 in EC, we examined the expression of FANCD2 in human specimens and databases, and discussed the possible mechanism of carcinogenesis by in vitro assays. Immunohistochemistry results showed overexpression of FANCD2 was detected in EC tissues compared to normal and atypical hyperplasia endometrium. Higher FANCD2 expression was correlated with deeper myometrial invasion (MI) and proficient mismatch repair status. The Cancer Genome Atlas (TCGA) database analysis showed FANCD2 was upregulated in EC compared with normal tissue. The high expression of FANCD2 was associated with poor overall survival in EC. Knockdown of FANCD2 expression in EC cell lines inhibited malignant proliferation and migration ability. We demonstrated that decreased FANCD2 expression results in increased DNA damage and decreased S-phase cells, leading to a decrease in proliferative capacity in EC cells. Down-regulated FANCD2 confers sensitivity of EC cells to interstrand crosslinking agents. This study provides evidence for the malignant progression and prognostic value of FANCD2 in EC. 10.1016/j.yexcr.2022.113388
Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination. The EMBO journal Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (I D2). Here, we present a 4.1 Å cryo-EM structure of I D2 complex bound to double-stranded DNA. We show that this complex, like ID2 and I D2 , is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the I D2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2 -DNA complex. The I D2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2 -DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an I D2 -DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, I D2 complex. 10.15252/embj.2022111898
LncRNA SNHG1 upregulates FANCD2 and G6PD to suppress ferroptosis by sponging miR-199a-5p/3p in hepatocellular carcinoma. Drug discoveries & therapeutics Ferroptosis is a form of regulated cell death (RCD) triggered by iron-dependent lipid peroxidation and is closely associated with the occurrence and progression of hepatocellular carcinoma (HCC). The lncRNA SNHG1 (small nucleolar RNA host gene 1) has been shown to play an oncogenic role in HCC, but its function in RCD other than autophagy and apoptosis is still unknown. Here, we investigated the correlation between SNHG1 and 156 typical markers of five RCD types based on RNA sequencing data from The Cancer Genome Atlas database and showed the negative regulators of ferroptosis FANCD2 (Fanconi anemia complementation group D2) and G6PD (glucose-6-phosphate dehydrogenase) to be the most highly and fifth most highly correlating factors with SNHG1, respectively. A competitive endogenous RNA network of SNHG1 - miR-199a-5p/3p - FANCD2/G6PD was constructed bioinformatically. In vitro experiments showed that overexpression of the miR-199a precursor led to a decrease in expression of SNHG1, FANCD2, and G6PD, whereas knockdown of SNHG1 decreased expression of FANCD2 and G6PD but increased levels of miR-199a-5p and miR-199a-3p in HCC cells (Huh7 and HepG2). In addition, knockdown of SNHG1 increased erastin-mediated ferroptosis, iron accumulation, and lipid peroxidation. These results suggest that SNHG1 upregulates FANCD2 and G6PD by sponging miR-199a, thereby inhibiting ferroptosis in HCC. Moreover, a signature based on expression of SNHG1, FANCD2, and G6PD was identified as being associated with overall survival and the immunological microenvironment in HCC. Collectively, this study identified the SNHG1-miR-199a-FANCD2/G6PD axis in HCC, which is a potential marker for the prognosis and therapy of this tumor. 10.5582/ddt.2023.01035
The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination. Nature structural & molecular biology DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2-FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2-FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2-FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2-FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2-FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2-FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair. 10.1038/s41594-022-00820-9
FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells. Communications biology Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy. 10.1038/s42003-022-04360-2
The ubiquitination machinery of the Fanconi Anemia DNA repair pathway. Progress in biophysics and molecular biology The Fanconi Anemia (FA) pathway maintains genome stability by preventing DNA damage from occurring when replication is blocked. Central to the FA pathway is the monoubiquitination of FANCI-FANCD2 mediated by a ubiquitin RING-E3 ligase complex called the FA core complex. Genetic mutation in any component of the FA core complex results in defective FANCI-FANCD2 monoubiquitination and phenotypes of DNA damage sensitivity, birth defects, early-onset bone marrow failure and cancer. Here, we discuss the mechanisms of the FA core complex and FANCI-FANCD2 monoubiquitination at sites of blocked replication and review our current understanding of the biological functions of these proteins in replication fork protection. 10.1016/j.pbiomolbio.2020.09.009
FANCD2 inhibits ferroptosis by regulating the JAK2/STAT3 pathway in osteosarcoma. BMC cancer BACKGROUND:This research aimed to investigate the roles of fanconi anemia complementation group D2 (FANCD2) on the regulation of ferroptosis in osteosarcoma progression. METHODS:The function of FANCD2 on cell viability, invasion, migration, and tumor growth were explored. FANCD2 and pathway-related genes were determined by western blot. Ferroptosis-associated markers were determined, including lipid peroxidation, labile iron pool (LIP), ferrous iron (Fe), and ferroptosis-related genes. RESULTS:FANCD2 expression was increased in osteosarcoma cells. FANCD2 knockdown reduced cell viability, invasion, and migration of osteosarcoma cells. FANCD2 knockdown regulated ferroptosis-related gene expression, and distinctly increased the levels of LIP, Fe, and lipid peroxidation, and these effects were reversed by a ferroptosis inhibitor Fer-1. In addition, JAK2 and STAT3 expression were reduced by silencing of FANCD2, and STAT3 activator (colivelin) distinctly reversed tumor suppressor effects of FANCD2 silencing on osteosarcoma development. CONCLUSION:These findings suggested that FANCD2 silencing could suppress osteosarcoma cell viability, migration, invasion, and tumor growth, and induced ferroptosis by regulating the JAK2/STAT3 axis. These findings may provide novel therapeutic ideas for clinical treatment of osteosarcoma. 10.1186/s12885-023-10626-7
FANCD2 and HES1 suppress inflammation-induced PPARɣ to prevent haematopoietic stem cell exhaustion. British journal of haematology The Fanconi anaemia protein FANCD2 suppresses PPAR to maintain haematopoietic stem cell's (HSC) function; however, the underlying mechanism is not known. Here we show that FANCD2 acts in concert with the Notch target HES1 to suppress inflammation-induced PPAR in HSC maintenance. Loss of HES1 exacerbates FANCD2-KO HSC defects. However, deletion of HES1 does not cause more severe inflammation-mediated HSC defects in FANCD2-KO mice, indicating that both FANCD2 and HES1 are required for limiting detrimental effects of inflammation on HSCs. Further analysis shows that both FANCD2 and HES1 are required for transcriptional repression of inflammation-activated PPARg promoter. Inflammation orchestrates an overlapping transcriptional programme in HSPCs deficient for FANCD2 and HES1, featuring upregulation of genes in fatty acid oxidation (FAO) and oxidative phosphorylation. Loss of FANCD2 or HES1 augments both basal and inflammation-primed FAO. Targeted inhibition of PPAR or the mitochondrial carnitine palmitoyltransferase-1 (CPT1) reduces FAO and ameliorates HSC defects in inflammation-primed HSPCs deleted for FANCD2 or HES1 or both. Finally, depletion of PPARg or CPT1 restores quiescence in these mutant HSCs under inflammatory stress. Our results suggest that this novel FANCD2/HES1/PPAR axis may constitute a key component of immunometabolic regulation, connecting inflammation, cellular metabolism and HSC function. 10.1111/bjh.17230
Phosphorylation by ATR triggers FANCD2 chromatin loading and activates the Fanconi anemia pathway. Cell reports The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes. 10.1016/j.celrep.2023.112721
ATM depletion induces proteasomal degradation of FANCD2 and sensitizes neuroblastoma cells to PARP inhibitors. BMC cancer BACKGROUND:Genomic alterations, including loss of function in chromosome band 11q22-23, are frequently observed in neuroblastoma, which is the most common extracranial childhood tumour. In neuroblastoma, ATM, a DNA damage response-associated gene located on 11q22-23, has been linked to tumorigenicity. Genetic changes in ATM are heterozygous in most tumours. However, it is unclear how ATM is associated with tumorigenesis and cancer aggressiveness. METHODS:To elucidate its molecular mechanism of action, we established ATM-inactivated NGP and CHP-134 neuroblastoma cell lines using CRISPR/Cas9 genome editing. The knock out cells were rigorously characterized by analyzing proliferation, colony forming abilities and responses to PARP inhibitor (Olaparib). Western blot analyses were performed to detect different protein expression related to DNA repair pathway. ShRNA lentiviral vectors were used to knockdown ATM expression in SK-N-AS and SK-N-SH neuroblastoma cell lines. ATM knock out cells were stably transfected with FANCD2 expression plasmid to over-expressed the FANCD2. Moreover, knock out cells were treated with proteasome inhibitor MG132 to determine the protein stability of FANCD2. FANCD2, RAD51 and γH2AX protein expressions were determined by Immunofluorescence microscopy. RESULTS:Haploinsufficient ATM resulted in increased proliferation (p < 0.01) and cell survival following PARP inhibitor (olaparib) treatment. However, complete ATM knockout decreased proliferation (p < 0.01) and promoted cell susceptibility to olaparib (p < 0.01). Complete loss of ATM suppressed the expression of DNA repair-associated molecules FANCD2 and RAD51 and induced DNA damage in neuroblastoma cells. A marked downregulation of FANCD2 expression was also observed in shRNA-mediated ATM-knockdown neuroblastoma cells. Inhibitor experiments demonstrated that the degradation of FANCD2 was regulated at the protein level through the ubiquitin-proteasome pathway. Reintroduction of FANCD2 expression is sufficient to reverse decreased proliferation mediated by ATM depletion. CONCLUSIONS:Our study revealed the molecular mechanism underlying ATM heterozygosity in neuroblastomas and elucidated that ATM inactivation enhances the susceptibility of neuroblastoma cells to olaparib treatment. These findings might be useful in the treatment of high-risk NB patients showing ATM zygosity and aggressive cancer progression in future. 10.1186/s12885-023-10772-y
Focal Point of Fanconi Anemia Signaling. International journal of molecular sciences Among human genetic diseases, Fanconi Anemia (FA) tops all with its largest number of health complications in nearly all human organ systems, suggesting the significant roles played by FA genes in the maintenance of human health. With the accumulated research on FA, the encoded protein products by FA genes have been building up to the biggest cell defense signaling network, composed of not only 22+ FA proteins but also ATM, ATR, and many other non-FA proteins. The FA D2 group protein (FANCD2) and its paralog form the focal point of FA signaling to converge the effects of its upstream players in response to a variety of cellular insults and simultaneously with downstream players to protect humans from contracting diseases, including aging and cancer. In this review, we update and discuss how the FA signaling crucially eases cellular stresses through understanding its focal point. 10.3390/ijms222312976
Exploring the Role of Mutations in Fanconi Anemia Genes in Hereditary Cancer Patients. Del Valle Jesús,Rofes Paula,Moreno-Cabrera José Marcos,López-Dóriga Adriana,Belhadj Sami,Vargas-Parra Gardenia,Teulé Àlex,Cuesta Raquel,Muñoz Xavier,Campos Olga,Salinas Mónica,de Cid Rafael,Brunet Joan,González Sara,Capellá Gabriel,Pineda Marta,Feliubadaló Lídia,Lázaro Conxi Cancers Fanconi anemia (FA) is caused by biallelic mutations in FA genes. Monoallelic mutations in five of these genes ( and ) increase the susceptibility to breast/ovarian cancer and are used in clinical diagnostics as bona-fide hereditary cancer genes. Increasing evidence suggests that monoallelic mutations in other FA genes could predispose to tumor development, especially breast cancer. The objective of this study is to assess the mutational spectrum of 14 additional FA genes ( and ) in a cohort of hereditary cancer patients, to compare with local cancer-free controls as well as GnomAD. A total of 1021 hereditary cancer patients and 194 controls were analyzed using our next generation custom sequencing panel. We identified 35 pathogenic variants in eight genes. A significant association with the risk of breast cancer/breast and ovarian cancer was found for carriers of mutations (odds ratio (OR) = 3.14 95% confidence interval (CI) 1.4-6.17, = 0.003). Two patients with early-onset cancer showed a pathogenic FA variant in addition to another germline mutation, suggesting a modifier role for FA variants. Our results encourage a comprehensive analysis of FA genes in larger studies to better assess their role in cancer risk. 10.3390/cancers12040829
FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability. Nucleic acids research FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea-induced stalled forks. The RAD51 recombinase has also been implicated in regulation of resection at stalled replication forks. The mechanistic contributions of these proteins to fork protection are not well understood. Here, we used purified FANCD2 and RAD51 to study how each protein regulates DNA resection at stalled forks. We characterized three mechanisms of FANCD2-mediated fork protection: (1) The N-terminal domain of FANCD2 inhibits the essential DNA2 nuclease activity by directly binding to DNA2 accounting for over-resection in FANCD2 defective cells. (2) Independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit multiple nucleases, including DNA2, MRE11 and EXO1. (3) Unexpectedly, we uncovered a new FANCD2 function: by stabilizing RAD51 filaments, FANCD2 acts to stimulate the strand exchange activity of RAD51. Our work biochemically explains non-canonical mechanisms by which FANCD2 and RAD51 protect stalled forks. We propose a model in which the strand exchange activity of FANCD2 provides a simple molecular explanation for genetic interactions between FANCD2 and BRCA2 in the FA/BRCA fork protection pathway. 10.1093/nar/gkad624
Mechanism, specificity, and function of FANCD2-FANCI ubiquitination and deubiquitination. The FEBS journal Fanconi anemia (FA) is a rare genetic disorder caused by mutations in any of the currently 22 known FA genes. The products of these genes, along with other FA-associated proteins, participate in a biochemical pathway, known as the FA pathway. This pathway is responsible for the repair of DNA interstrand cross-links (ICL) and the maintenance of genomic stability in response to replication stress. At the center of the pathway is the monoubiquitination of two FA proteins, FANCD2 and FANCI, on two specific lysine residues. This is achieved by the combined action of the UBE2T ubiquitin-conjugating enzyme and a large multicomponent E3 ligase, known as the FA-core complex. This E2-E3 pair specifically targets the FANCI-FANCD2 heterodimer (ID2 complex) for ubiquitination on DNA. Deubiquitination of both FANCD2 and FANCI, which is also critical for ICL repair, is achieved by the USP1-UAF1 complex. Recent work suggests that FANCD2 ubiquitination transforms the ID2 complex into a sliding DNA clamp. Further ubiquitination on FANCI does not alter this closed-on-DNA ID2 conformation. However, the resulting dimonoubiquitinated complex is highly resistant to USP1-UAF1 deubiquitination. This review will provide an update on recent work focusing on how specificity in FANCD2 ubiquitination and deubiquitination is achieved. Recent findings shedding light to the mechanisms, molecular functions, and biological roles of FANCI/FANCD2 ubiquitination and deubiquitination will be also discussed. ENZYMES: UBA1 (, UBE2T (, FANCL (, USP1 ( 10.1111/febs.16077
Structural insight into FANCI-FANCD2 monoubiquitination. Li Landing,Tan Winnie,Deans Andrew J Essays in biochemistry The Fanconi anemia (FA) pathway coordinates a faithful repair mechanism for DNA damage that blocks DNA replication, such as interstrand cross-links. A key step in the FA pathway is the conjugation of ubiquitin on to FANCD2 and FANCI, which is facilitated by a large E3 ubiquitin ligase complex called the FA core complex. Mutations in FANCD2, FANCI or FA core complex components cause the FA bone marrow failure syndrome. Despite the importance of these proteins to DNA repair and human disease, our molecular understanding of the FA pathway has been limited due to a deficit in structural studies. With the recent development in cryo-electron microscopy (EM), significant advances have been made in structural characterization of these proteins in the last 6 months. These structures, combined with new biochemical studies, now provide a more detailed understanding of how FANCD2 and FANCI are monoubiquitinated and how DNA repair may occur. In this review, we summarize these recent advances in the structural and molecular understanding of these key components in the FA pathway, compare the activation steps of FANCD2 and FANCI monoubiquitination and suggest molecular steps that are likely to be involved in regulating its activity. 10.1042/EBC20200001